The Third Hydrogenase: More Natural Organometallics

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Organometallics 2010, 29, 6146–6156 DOI: 10.1021/om1008567

The Third Hydrogenase: More Natural Organometallics Joseph A. Wright, Peter J. Turrell, and Christopher J. Pickett* Energy Materials Laboratory, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom Received September 3, 2010

The [Fe]-hydrogenase, also known as “H2-forming methylenetetrahydromethanopterin dehydrogenase” (Hmd) or “iron-sulfur cluster-free hydrogenase”, is the third distinct class of hydrogenase enzyme to be discovered. In the presence of its carbocation substrate, it catalyzes the reversible cleavage of dihydrogen into Hþ and H-, placing the later concomitantly onto the substrate molecule. This review describes the emerging chemistry behind this fascinating enzyme.

Introduction The redox-active [NiFe]- and [FeFe]-hydrogenase enzymes are capable of catalyzing the reversible interconversion of dihydrogen with protons and electrons. They do this at such high rates that an understanding of their chemistry has the potential to impact technology for energy transduction in a “hydrogen economy”.1 In the last 10 years or so much research effort has been targeted at experimentally and theoretically modeling the structure and function of the organometallic active sites of these enzymes (Figure 1). There is a third hydrogenase which contains a mononuclear organometallic site,2 “H2-forming methylenetetrahydromethanopterin dehydrogenase” (Hmd) or [Fe]-hydrogenase (also infrequently known as “iron-sulfur cluster-free hydrogenase”). All three hydrogenases are phylogenetically distinct, but whereas the [NiFe]- and [FeFe]-hydrogenases are redox active and catalyze the same interconversion, [Fe]hydrogenase is not redox active and performs different chemistry. This third hydrogenase catalyzes the heterolytic cleavage of dihydrogen only in the presence of its substrate, N5,N10-methenyltetrahydromethanopterin (methenyl-H2MPT) (Scheme 1). The enzyme was initially believed to be metalfree.3-5 The presence of a redox-inactive iron center in the enzyme was first reported in 2004,5 with crystallographic evidence for the nature of the active site being reported in 20086 and revised in 2009.7 In 2007, Shima and Thauer reviewed biological aspects of [Fe]-hydrogenase.4 In this review we focus on the emerging chemistry of the active site on the basis of structural and (1) Tard, C.; Pickett, C. J. Chem. Rev. 2009, 109, 2245–2274. (2) Zirngibl, C.; Hedderich, R.; Thauer, R. K. FEBS Lett. 1990, 261, 112–116. (3) Thauer, R. K.; Klein, A. R.; Hartmann, G. C. Chem. Rev. 1996, 96, 3031–3042. (4) Shima, S.; Thauer, R. K. Chem. Rec. 2007, 7, 37–46. (5) Lyon, E. J.; Shima, S.; Burrman, G.; Chowdhuri, S.; Batschauer, A.; Steinbach, K.; Thauer, R. K. Eur. J. Biochem. 2004, 271, 195–204. (6) Shima, S.; Pilak, O.; Vogt, S.; Schick, M.; Stagni, M. S.; MeyerKlaucke, W.; Warkentin, E.; Thauer, R. K.; Ermler, U. Science 2008, 321, 572–575. (7) Hiromoto, T.; Ataka, K.; Pilak, O.; Stagni, M. S.; Meyer-Klaucke, W.; Warkentin, E.; Thauer, R. K.; Shima, S.; Ermler, U. FEBS Lett. 2009, 583, 585–590. pubs.acs.org/Organometallics

Published on Web 10/22/2010

Figure 1. Active sites of the [NiFe]- and [FeFe]-hydrogenases.

mechanistic data now available from the enzymatic and model systems.

Biological Aspects [Fe]-hydrogenase catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPTþ) with H2 to methylene-H4MPT and Hþ, which is an intermediary step in the reduction of CO2 to methane using H2 by methanogens under nickel-deficient conditions.8 The enzyme is difficult to extract and manipulate, as it is sensitive to dioxygen and light and is only expressed in large quantities when the producing strain is grown under nickel-limiting and red-light conditions.5 The availability of high-purity enzyme material for detailed study using biophysical techniques5-7,9-13 allowed its identification as a metalloenzyme, and subsequently our understanding of structure and function has dramatically expanded in the last 4 years. Efforts to identify the nature of the metal center using infrared9 and extended X-ray absorption fine structure (EXAFS)10 methods disclosed the presence of two carbonyl ligands on the metal in a cis arrangement. M€ ossbauer spectroscopy indicated that the (8) Thauer, R. K. Microbiology 1998, 144, 2377–2406. (9) Lyon, E. J.; Shima, S.; Boecher, R.; Thauer, R. K.; Grevels, F.-W.; Bill, E.; Roseboom, W.; Albracht, S. P. J. J. Am. Chem. Soc. 2004, 126, 14239–14248. (10) Korbas, M.; Vogt, S.; Meyer-Klaucke, W.; Bill, E.; Lyon, E. J.; Thauer, R. K.; Shima, S. J. Biol. Chem. 2006, 281, 30804–30813. (11) Shima, S.; Lyon, E. J.; Thauer, R. K.; Mienert, B.; Bill, E. J. Am. Chem. Soc. 2005, 127, 10430–10435. (12) Shima, S.; Lyon, E. J.; Sordel-Klippert, M.; Kauss, M.; Kahnt, J.; Thauer, R. K.; Steinbach, K.; Xie, X.; Verdier, L.; Griesinger, C. Angew. Chem. 2004, 116, 2601–2605. Angew. Chem., Int. Ed. 2004, 43, 2547-2551. (13) Pilak, O.; Mamat, B.; Vogt, S.; Hagemeier, C. H.; Thauer, R. K.; Shima, S.; Vonrhein, C.; Warkentin, E.; Ermler, U. J. Mol. Biol. 2006, 358, 798–809. r 2010 American Chemical Society

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Scheme 1. Stereospecific Reversible Hydride Transfer to N 5,N10-Methenyltetrahydromethanopterin (1), Catalyzed by the [Fe]-Hydrogenase

Figure 2. Pathway for formation of the light-inactivated cofactor derivative.12

Figure 3. Light-inactivated cofactor derivative as isolated from M. marburgensis.12

iron center in the active site must be either low-spin Fe(0) or Fe(II), with the latter more likely argued, as the enzyme reversibly binds cyanide ions.11 The iron cofactor could be removed from the enzyme and then exposed to light to yield a light-inactivated cofactor derivative (Figure 2).12 A combination of multinuclear NMR techniques and mass spectroscopy was used to indentify this organic molecule as 3, with the binding site for iron presumed to be the pyridone nitrogen (Figure 3). Photolability of CO from a metal center is of course a relatively common reaction, and such loss of CO from the iron site must be an early step in the release of the metal center from the organic cofactor ligand. The full detail of the active site has subsequently been revealed by X-ray crystallography. Crystallization of the apoenzyme showed that the enzyme is a homodimer which can be subdivided into a central globular unit attached to two peripheral units in a linear manner. Each of the peripheral units corresponds to the N-terminal domain of one subunit and is composed of an R/β structure that belongs to the Rossmann fold protein family.13 The central globular unit is composed of the interlocking helices of the C-terminal segment of both subunits. Crystallization of the apoenzyme from M. jannaschii with the iron-containing cofactor (FeGP) produced under redlight conditions and in the absence of oxygen led to the first crystal structure of the holoenzyme (Figure 4).6 The metal binding site was initially modeled using the carboxylic acid containing pyridinol 3 (Figure 5 (left)). In this original model

the pyridinol hydroxy group is located close to a metalbound carbonyl, while the pendant carboxylic acid group is close to the “solvent” site on the metal. Subsequently, crystallization of a mutant enzyme in which cysteine 176 is mutated revealed that this binding mode was unlikely.7 The mutant enzyme was crystallized in the presence of DTT (2S,3S-dithiothreitol), which binds to the metal center through both a sulfur and an oxygen (Figure 6). The second hydroxy group of DTT showed a severe steric interaction with the postulated position for the pendant CO2H group of the cofactor. Rotating the pyridinol ring by 180° around the Fe-N bond and replacing the pendant acid group by an acyl ligand avoided this clash. The crystal structure for the holoenzyme was therefore rerefined using the same modification of the cofactor (Figure 4, Figure 5 (right)).7 This revised structure is fully consistent with the earlier spectroscopic data for the active site of the enzyme and is also in accord with recent theoretical work which supports the formulation as an iron(II) complex bound by two carbonyls, a cysteinyl sulfur, acylpyridine, and a sixth oxygen-containing ligand.14 Prior to the availability of a crystal structure, EXAFS data for the enzyme had been interpreted in terms of a four- or five-coordinate low-valent iron center.10 The data were rerefined as part of the revision of the crystal structure and are fully concordant with the new formulation.7 There is good agreement between the metal-ligand bond distances given by the two techniques.

Synthetic Models Attempts to model aspects of the [Fe]-hydrogenase active site began before the crystal structure was reported, targeting elements of the structure as proposed on the basis of spectroscopic data. Rauchfuss and co-workers modeled the lightinactivated cofactor derivative 3 using the known ligand 4 (Figure 7). This was used to construct model 5, in which ruthenium acts as a surrogate for iron.15 While this model lacks carbonyl and sulfur ligands, it does possess a pyridinol group, which must prevent κ2N,O chelation by the stronger metal binding affinity of the carboxylate group. Before the availability of X-ray diffraction data, spectroscopic techniques had revealed the presence of a cis arrangement of carbonyl ligands in the [Fe]-hydrogenase active site, along with a sulfur ligand.9,10 The infrared spectrum of the [Fe]-hydrogenase shows two absorptions for the carbonyl groups at 1972 and 2031 cm-1 in water, with the two bands having approximately equal intensity.9 For two carbonyl (14) Nakatani, N.; Nakao, Y.; Sato, H.; Sakaki, S. Chem. Lett. 2009, 38, 958–959. (15) Royer, A. M.; Rauchfuss, T. B.; Wilson, S. R. Inorg. Chem. 2008, 47, 395–397.

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Figure 4. Crystal structure of the wild-type holoenzyme from Methanocaldococcus jannaschii, showing the two protein subunits (cyan and green) and the bound FeGP cofactor (gray, carbon; blue, nitrogen; red, oxygen; orange, phosphorus; yellow, sulfur; dark brown, iron).6 The structure of the apoenzyme shows the same homodimer arrangement but contains a “closed” cleft between the peripheral and central units.13

Figure 5. Originally proposed (left) and revised (right) models for the active site of the [Fe]-hydrogenase.6,7 Note the rotation of pyridinol orientation about the Fe-N bond between the original and revised models.

Figure 7. Pyridinol-containing model (Cp* = C5Me5).15

Figure 8. Iron(I) cis-dicarbonyl complex reported by Holland and co-workers (Ar = 2,6-(i-Pr)2C6H3).17

Figure 6. (a) Crystal structure of the mutant enzyme crystallized in the presence of DTT, showing a single protein subunit and omitting the part of the protein not surrounding the FeGP cofactor (color scheme as in Figure 4).7 (b) Illustration of the binding motif of the iron center, highlighting the DTT molecule in red. Both oxygen atoms in the DTT molecule are potentially involved in hydrogen bonding with the hydroxy group of the pyridinol.

groups with the same force constant, the angle 2θ between the two is given by the formula Isym/Iasym = cotan2 θ, where Isym and Iasym are the infrared intensities for the symmetric (16) Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry, 5th ed.; Wiley: Chichester, U.K., 1988; pp 1035-1037.

and antisymmetric stretches, respectively.16 The infrared data for the enzyme therefore strongly suggest an angle between the two carbonyls of approximately 90° in solution. Early modeling studies concentrated on reproducing this cis arrangement, with other supporting ligands varied as necessary to obtain the desired structures, although the inclusion of at least one sulfur donor has been a feature of most of the [Fe]-hydrogenase models reported in the literature. However, it should be noted that infrared data similar to those for the natural system can be obtained for iron(II) complexes utilizing a wide range of ancillary ligands of more or less relevance to the biological system. In the earlier work, a low-coordination-number iron center bearing two carbonyl ligands had been suggested on the basis of EXAFS data for the enzyme (vide supra).10 Holland and co-workers modeled this ligand arrangement in the fourcoordinate complex 6 (Figure 8), with carbonyl stretches at 1917 and 1996 cm-1.17 Holland noted that 6 is an iron(I) complex, whereas the enzyme features an iron(II) metal center. Koch and co-workers synthesized [NiFe]- and [Fe]-hydrogenase subsite models bearing a bis(thiolato)phosphine, (17) Sadique, A. R.; Brennessel, W. W.; Holland, P. L. Inorg. Chem. 2008, 47, 784–786.

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Scheme 2. cis-Dicarbonyl Models Reported by Koch and Co-workers18

Figure 9. Models used for XANES studies by Salome-Stagni et al.20 Scheme 4. Synthesis of Pyridone-Containing Model (Ar = 2,6-Me2C6H3)21

Scheme 3. Synthesis of Model Compound for NVRS studies As Reported by Rauchfuss and Co-workers19

Scheme 5. Synthesis of a Phosphine-Free Model by Wang et al.22

starting from the tricarbonyl system 7 (Scheme 2).18 This initial model can be converted to a cis-dicarbonyl by the action of cyanide ions, with the first-formed product 8a isomerizing to 8b when exposed to ambient light. From the infrared spectra of these complexes Koch and co-workers suggested that the [Fe]-hydrogenase active site is similar in electron density to 8b, but in a lower symmetry environment. Rauchfuss and co-workers reported compound 9 (Scheme 3) as part of a study of the nuclear resonance vibrational spectroscopy (NVRS) spectrum of the [Fe]-hydrogenase.19 This molecule again exhibits the cis-dicarbonyl arrangement found in the natural system, but with the presence of two abiological phosphine ligands and with a chelating dithiolate group present. This molecule exhibits two IR stretches in toluene solution, at 1939 and 1998 cm-1, while in the solid state there are a larger number of stretches in the same region (1999, 1984, 1950, 1933, and 1910 cm-1). The authors attributed this phenomenon to isomerization of 9, with a single cis isomer present in toluene solutions but multiple species in the solid state. The NVRS measurements were used to define a minimal ligand set for the [Fe]-hydrogenase, namely two cis carbonyls, a sulfur, and a light atom, suggested to be the pyridyl cofactor nitrogen. In order to understand the X-ray absorption near edge spectroscopy (XANES) spectrum of the [Fe]-hydrogenase, Salome-Stagni et al. synthesized a number of simple model (18) Melgarejo, D. Y.; Chiarella, G. M.; Koch, S. A. Synthetic analogs for the iron centers in Ni-Fe and the iron-sulfur cluster free hydrogenase enzymes. Abstracts of Papers; 234th National Meeting of the American Chemical Society, Boston, MA, United States, August 19-23, 2007; American Chemical Society: Washington, DC, 2007; http://oasys2. confex.com/acs/234nm/techprogram/P1116341.HTM. (19) Guo, Y.; Wang, H.; Xiao, Y.; Vogt, S.; Thauer, R. K.; Shima, S.; Volkers, P. I.; Rauchfuss, T. B.; Pelmenschikov, V.; Case, D. A.; Alp, E. E.; Sturhahn, W.; Yoda, Y.; Cramer, S. P. Inorg. Chem. 2008, 47, 3969–3977.

compounds bearing carbonyl ligands (Figure 9).20 A comparison of the XANES data from these simple model complexes with those for the native enzyme supported the formulation of the active site as containing Fe(II) with octahedral geometry and with either five or six donor atoms present. The inclusion in model compounds of a nitrogen-sulfur chelate has been used as a successful approach to generate ligand sets which include more of the features of the natural active site than simply the cis-dicarbonyl arrangement. The importance of chelation to the pyridine group has been demonstrated by Hu and colleagues, who have included a pyridone molecule in a cis-dicarbonyl system (Scheme 4).21 In contrast to the natural system, the simple pyridone forms a nitrogen-oxygen chelate, with carbonyl bands at 1987 and (20) Salome-Stagni, M.; Stellato, F.; Whaley, C. M.; Vogt, S.; Morante, S.; Shima, S.; Rauchfuss, T. B.; Meyer-Klaucke, W. Dalton Trans. 2010, 39, 3057–3064. (21) Obrist, B. V.; Chen, D.; Ahrens, A.; Sch€ unemann, V.; Scopelliti, R.; Hu, X. Inorg. Chem. 2009, 48, 3514–3516.

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2032 cm-1. Reaction of 13 with a bulky sodium thiolate leads to the formation of the thiolate-containing system 14, as indicated by multinuclear NMR, IR, and elemental analysis. Liu, Pickett, and co-workers reported a tetradentate ligand containing both pyridine and thiolate donors and used this to generate a model complex featuring the cis arrangement of carbonyls without the need for abiological ancillary phosphines (Scheme 5).22 Model 15 exhibits two IR bands in the carbonyl region at 1973 and 2026 cm-1, in very good agreement with those observed in the natural system. Directly bonding a sulfur atom onto a pyridine is a strategy that has produced a number of model compounds. This approach was reported by Halder et al. in the synthesis of a molecule also containing a nucleophilic carbene ligand (16, Figure 10),23 but this system is limited in that it does not possess any carbonyl ligands. The same ligand was used by Liaw and co-workers in a model originally designed to mimic one iron center in the [FeFe]-hydrogenase (Figure 11).24 This compound does feature the desired cis-dicarbonyl arrangement, with infrared stretches at 1989 and 2040 cm-1. The same ligand system was also exploited by Darensbourg and co-workers to give carbonyl-containing models for M€ ossbauer spectroscopy and infrared studies (Figure 12).25 The IR data from a large number of models was examined (Table 1), showing that good agreement with the natural system was only seen when the model included the chelating ligand. This is particularly notable when comparing with the pyridine complex (22; Table 1, entry 6), which is a much poorer model for the active site. Clearly the inclusion of a thiolate group is significant in obtaining a high-quality model. M€ ossbauer data for the model complexes gave best agreement with the native active site for the tricarbonyl complex 22. Darensbourg and co-workers point out that this calls into question the usefulness of M€ ossbauer data in assigning the structure of biological molecules: it is clear that 22 is a much poorer structural model for the active site than 25.

Both Liaw and Darensbourg have extended the extended the concept of a chelating nitrogen-sulfur ligand. As part of a study into [FeFe]-hydrogenase models, Liaw and co-workers synthesized 26 (Figure 13).26 This features an aniline-derived thiol in place of the pyridine-derived ligand in 17. As an anionic model, the CO stretches for this complex (at 1929 and 1992 cm-1) are unsurprisingly shifted from those of the [Fe]-subsite and neutral model complexes. Darensbourg and co-workers exploited the same aniline ligand, increasing the bite angle of the ligand to give overall less strained complexes (Scheme 6, Table 1).27 These complexes are five-coordinate, leaving a vacant site which can be viewed as similar to the “solvent” site in the [Fe]-hydrogenase. Only 27a shows any tendency to bind carbon monoxide under ambient conditions. Protonation of 27a with the strong acid HBF4 3 Et2O under a CO atmosphere leads to the formation of a new species with IR stretches at 2046 and 2102 cm-1, which was assigned as the meridional tricarbonyl. Treatment of this species with base regenerates 27a. The third challenge in modeling the [Fe]-hydrogenase site is to introduce the acyl ligand present in the natural system or a close structural analogue. Rauchfuss and co-workers approached this area by reaction of an iron(0) source with a thioester containing a phosphine for chelation (Scheme 7).28 The resulting oxidative addition reaction yields the desired acyl ligation of the metal along with a bound thiolate in an elegant single step. The reaction product from this process is the tricarbonyl 28, which will reversibly lose a carbon monoxide molecule to form the dimeric structure 29. Compound 28 shows carbonyl stretches at 1981, 2020, and 2075 cm-1, very similar to the bands observed for the CO-inhibited form of the enzyme (2001, 2025, and 2074 cm-1). The dimer 29 exists as two isomers (axial-equatorial and equatorial-axial), which interconvert on the time scale of hours in solution, as indicated by 31P NMR. It can be converted back to 28 under a carbon monoxide atmosphere after extended periods, and this route gives high-purity 28. Compound 28 also reacts with cyanide to yield 30, which exhibits bands very similar to those for the cyanide-inhibited form of the enzyme (1954, 2013, and 2094 cm-1 for 30 vs 1956, 2020, and 2090 cm-1 for the CN-inhibited enzyme). Hu and co-workers have taken a different approach to introduction of an acyl group, with the acyl ligand not part of a chelating system (Scheme 8).29 Starting from the acylcontaining precursor 31, reaction with the sodium salt of 2-mercapto-6-methylpyridine gives the dimeric product 32. This can be cleaved into monomeric units by the action of a number of donor ligands. Particularly notable are the reactions with cyanide and carbon monoxide, which yield compounds 33c,d, respectively. These are good structural models for the [Fe]-hydrogenase active site, containing no abiological groups and mimicking the majority of the features of the active site. However, it is notable that they contain a relatively “tight” nitrogen-sulfur chelate, in contrast to the natural system, where the sulfur is not bound to the nitrogen ligand and where there are no small bite angles.

(22) Wang, X.; Li, Z.; Zeng, X; Luo, Q.; Evans, D. J.; Pickett, C. J.; Liu, X. Chem. Commun. 2008, 45, 3555–3557. (23) Halder, P.; Dey, A.; Paine, T. K. Inorg. Chem. 2009, 48, 11501– 11503. (24) Liaw, W.-F.; Chen, C.-H.; Lee, G. H.; Peng, S.-M. Organometallics 1998, 17, 2370–2372. (25) Li, B.; Liu, T.; Popescu, C. V.; Biko, A.; Darensbourg, M. Y. Inorg. Chem. 2009, 48, 11283–11289.

(26) Liaw, W.-F.; Lee, N.-H.; Chen, C.-H.; Lee, C.-M.; Lee, G.-H.; Peng, S.-M. J. Am. Chem. Soc. 2000, 122, 488–494. (27) Liu, T.; Li, B.; Popescu, C. V.; Bilko, A.; Perez, L. M.; Hall, M. B.; Darensbourg, M. Y. Chem. Eur. J. 2010, 16, 3083–3089. (28) Royer, A. M.; Rauchfuss, T. B.; Gray, D. L. Organometallics 2009, 28, 3618–3620. (29) Chen, D.; Scopelliti, R.; Hu, X. J. Am. Chem. Soc. 2010, 132, 928–929.

Figure 10. Model precursor reported by Halder et al.23

Figure 11. Model using a chelating pyridine-thiolate ligand reported by Liaw and co-workers.24

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Figure 12. Model series synthesized by Darensbourg and co-workers.25 Table 1. Infrared Absorptions for a Series of Model Compounds Recorded in CH2Cl225,27 entry

compd

ν(CO)/cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

18a 18b 19 20 21 22 23 24a 24b 24c 25a 25b 27a 27b 27c Hmda

2018, 2040, 2087 2023, 2034, 2082 2019, 2039, 2082 2003, 2044 2002, 2043 2041, 2050, 2095 2022, 2047, 2093 2017, 2031, 2081 2032, 2045, 2093 2053, 2104 1971, 2019 1983, 2031 1927, 1985 1956,2012 1942, 2002 1944, 2011

a

Recorded in thf.

Figure 13. Model using a chelating aniline-thiolate ligand reported by Liaw and co-workers.26

Turrell et al. have recently reported the synthesis of a close structural mimic for the [Fe]-hydrogenase active site incorporating a chelating carbamoyl group as a surrogate for the acyl unit (Scheme 9).30 The use of carbonyl insertion starting from the readily available 2-aminopyridine yields the monomeric model complex 34. In this way, complex synthesis of the acyl group is avoided. Subsequent reaction with 2mercaptoethanol yields the dimeric molecule 35, in which the pendant alcohol does not chelate to the metal. However, addition of the bulky thiol 2,6-dimethylthiolphenol to 34 does lead to a monomeric thiolate-containing model (36). Here, the full coordination sphere seen in the [Fe]-hydrogenase is completed, with the carbamoyl as a good model for (30) Turrell, P. J.; Wright, J. A.; Peck, J. N. T.; Oganesyan, V. S.; Pickett, C. J. Angew. Chem., Int. Ed. 2010, 49, 7508-7511. (31) Chen, D.; Scopelliti, R.; Hu, X. Angew. Chem., Int. Ed. 2010, 49, 7512-7515.

Scheme 6. Chelating Nitrogen-Sulfur Carbonyls Used by Darensbourg and Co-workers27

the acyl group and the acetonitrile in place of the weakly bound water in the enzyme (Figure 14). The CO stretching frequencies for 36 (1958 and 2026 cm-1 in the solid state) agree closely with those for the enzyme (1972 and 2031 cm-1 in aqueous solution). Hu and co-workers have very recently accessed an acylmethylpyridine model for the [Fe]-hydrogenase (Scheme 10).31 Reaction of the iron(0) starting material Fe(CO)5 with a lithium picoline salt yields the acyl complex 37, which exhibits three infrared bands in the carbonyl region, suggesting trigonalbipyramidal geometry at iron with no nitrogen binding. Oxidation with iodine yields unstable iron(II) species, which can be reacted directly with sodium 6-methyl-2-mercaptopyridine to give the acyl complex 38, in which both pyridine and acyl groups are bound to the metal, as confirmed by X-ray crystallography for 38a. Comparison of the geometric data for 38a with those for 36 and the [Fe]-hydrogenase shows that all three are in close agreement with regard to bond distances (Figure 14). The κ2N,S chelate in 38a shows distortion from ideal octahedral geometry of approximately 20°, as seen in earlier models bearing this ligand. Both 38a and 38b exhibit two infrared stretches in the carbonyl region (1962 and 2029 cm-1 for 38a and 1961 and 2026 cm-1 for 38b). The acyl CO group in 38 is exchangeable with 13CO, and this reaction can be monitored over the course of 1 week by NMR spectroscopy. This strongly suggests that an elimination-addition mechanism is accessible for this system, which may well parallel the formation of 34 (vide supra). This reaction is accelerated by ultraviolet light, which also leads to isomerization of 38 to 380 , a process which is reversed in the dark (Scheme 11).

Mechanism Investigation of the mechanism of action for the enzyme began soon after the isolation of the purified [Fe]-hydrogenase

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Scheme 7. Thioester Activation As a Route to a [Fe]-Hydrogenase Modela,28

a

In 30, one of the CX and CY groups is CO and the second is CN.

Scheme 8. Generation of an Acyl-Containing Model by the Hu Group29

was reported. The [Fe]-hydrogenase does not catalyze the isotopic exchange between 3H2 (tritium gas) and 1H2O unless the substrate is present.32 Investigation of the kinetic isotope effects seen in the presence of labeled water confirmed that the reaction mechanism involves a hydride transfer to 1.33 Subsequently, two-dimensional NMR experiments using labeled materials were used to establish that the hydride transfer occurs stereospecifically to the pro-R face of 1 to form 2 (Scheme 1, pro-R hydrogen in red).34,35 These studies also established that the hydride is transferred from molecular hydrogen: i.e., that the enzyme catalyzes heterolytic cleavage of dihydrogen in the presence of 1. Studies using para hydrogen were used to conclusively demonstrate that (32) Zirngbl, C.; Van Dongen, W.; Schw€ orer, B.; Von B€ unau, R.; Richter, M.; Klein, A.; Thauer, R. K. Eur. J. Biochem. 1992, 208, 511– 520. (33) Schw€ orer, B.; Fernandez, V. M.; Zirngibl, C.; Thauer, R. K. Eur. J. Biochem. 1993, 212, 255–261. (34) Schleucher, J.; Griesinger, C.; Schw€ orer, B.; Thauer, R. K. Biochemistry 1994, 33, 3986–3993. (35) Scheucher, J.; Schw€ orer, B.; Thauer, R. K.; Griesinger, C. J. Am. Chem. Soc. 1995, 117, 2941–2942. (36) Hartmann, G. C.; Santamaria, E.; Fernndez, V. M.; Thauer, R. K. J. Biol. Inorg. Chem. 1996, 1, 445–450.

Scheme 9. Synthesis of Carbamoyl-Containing Models Reported by Pickett and Co-workers30

the heterolytic cleavage is dependent on activation by the substrate.36 Dihydrogen exists in two nuclear spin states, with the spins parallel (ortho) or antiparallel (para). Suitable preparation will produce dihydrogen in which the para state is enhanced. This enhancement is lost only slowly at room temperature but is rapidly depleted when the hydrogenhydrogen bond is cleaved.37 Para hydrogen enhancement is not reduced by enzyme or 1 but is reduced when the enzyme and substrate are combined. Reaction of D2 with the enzyme in H2O results in the parallel formation of HD and H2,36 meaning that HD cannot be an intermediate on the pathway to H2 formation. This has been rationalized in terms of a side-on binding arrangement in which koff = kexchange (Scheme 12). What does all of this mean? The active site of the enzyme cannot bear an exchangeable proton, as this would readily carry out isotopic scrabbling in the test molecules in the absence of the substrate. This is particularly notable, as the reaction of 1 is itself reversible. The para hydrogen experiments show that the activation of hydrogen is dependent on the substrate and that there cannot be a “sticky” intermediate in which the hydrogen-hydrogen bond is broken to give a nonexchanging species. There are three possible explanations for this substrate dependence. First, the substrate may (37) Vignais, P. M. Coord. Chem. Rev. 2005, 249, 1677–1690.

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Figure 14. Comparison of the geometric parameters in the [Fe]-hydrogenase active site (left)7 with those of 36 (middle)30 and 38a (right).31 The absolute stereochemistry of the model iron centers has been drawn to match that of the natural system. Scheme 10. Synthesis of an Acylmethylpyridine-Containing Model by the Hu Group (a, R = Me; b, R = OMe)31

Scheme 12. Mechanism To Explain Parallel Formation of H2 and HD from a Reaction Mixture Containing D2, H2O, the [Fe]Hydrogenase and 1a,36

Scheme 11. Possible Isomerization Route for 3831

a The metal center and substrate are represented by the red “Fe” and blue “C”, respectively.

itself be involved in activation of the dihydrogen molecule in a “push-pull” fashion in concert with the metal center (vide infra). Second, the dihydrogen binding site may be blocked by a hydrogen-bonding network which is disrupted on substrate binding, allowing access for dihydrogen and subsequent reaction. Third, activation may not involve the metal center at all, meaning that the substrate is critically relevant to activation. The unique nature of the iron complex in the [Fe]-hydrogenase suggests that this last explanation is perhaps rather unlikely. Before its identification as a metalloenzyme, Berkessel and Thauer proposed a reaction mechanism for the enzyme based on analogy with the intermediates observed under superacidic conditions.38 This model involves protonation of the already positively charged substrate to yield a highly reactive dicationic system (Figure 15). The concept was developed (38) Berkessel, A.; Thauer, R. K. Angew. Chem. 1995, 107, 24182421; Angew. Chem., Int. Ed. 1995, 34, 2247-2250. (39) Berkessel, A. Curr. Opin. Chem. Biol. 2001, 5, 486–490.

Figure 15. Potential dicationic intermediates proposed by Berkessel and Thauer.38

further by Berkessel,39 who postulated a metal-free activation mechanism in which suitably arranged δþ and δ- centers activate the dihydrogen molecule for hetetolytic cleavage. Although Berkessel’s mechanism for purely organic cleavage of H2 by the [Fe]-hydrogenase has been abandoned, there are attractive elements which resonate with a multicenter mechanism involving Fe(II), as developed below. Thauer and colleagues examined the rate of exchange between 3H2 and 1H2O catalyzed by the [Fe]-hydrogenase and the dependence of this rate on the presence of the substrate 1.40 This work confirmed the need for 1 to be present to observe significant reaction rates and raised the question of (40) Vogt, S.; Lyon, E. J.; Shima, S.; Thauer, R. K. J. Biol. Inorg. Chem. 2008, 13, 97–106.

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Scheme 13. Postulated Reaction Mechanism for the [Fe]-Hydrogenasea,41

a

The circles represent the enzyme scaffold, with the “open” conformer in yellow and the “closed” conformer in green.

Figure 16. Crystal structure of the enzyme mutated at cysteine 176 crystallized in the presence of DTT and 1 (color scheme as in Figure 1).41

the role of the iron center in the reaction mechanism. Thauer and co-workers highlight the fact that dihydrogen binding by the metal center in the absence of 1 must be weak (as there is essentially no reaction without 1 present) and that any mechanism must account for this and the activation of the system once the substrate is bound. Subsequently, Shima and (41) Hiromoto, T.; Warkentin, E.; Moll, J.; Ermler, U.; Shima, S. Angew. Chem. 2009, 121, 6579-6582; Angew. Chem., Int. Ed. 2009, 48, 6457-6460.

co-workers crystallized a mutant enzyme in the presence of DTT and the substrate (Figure 16).41 This structure reveals the enzyme in the “open” form, with the cleft between the central dimer section and the head unit relatively wide. The substrate is essentially planar in the crystal structure, in contrast to the NMR evidence, which suggests sp3 hybridization for the carbocationic portion. Shima and co-workers postulate that in the catalytically active enzyme closure of the cleft leads to conformational change in the substrate followed by side-on dihydrogen binding and turnover (Scheme 13). This mechanism is fully in accord with the available experimental data for the enzyme and points to a role for both the metal center and pyridinone ligand in dihydrogen activation. This mechanism can therefore be regarded as a development of that proposed by Berkessel and Thauer, where the carbocation acts as a hydride acceptor.38,39 While a metal is involved in activation, this is not in the classical activation role seen in noble-metalcatalyzed reactions. A key concept in this mechanistic proposal is the conformation change induced in the enzyme scaffold on substrate binding (illustrated by a color change from yellow to green in Scheme 13). The proposed analogy of the [Fe]-hydrogenase activation mechanism to that of superacidic solution38,39 inspired Murphy and co-workers to examine the reactivity of pyridinium salts (Scheme 14).42,43 Hydrogenation of the dicationic pyridinium salt 39 proceeds smoothly to yield 40, which cannot be hydrogenated further under the same conditions. This provides support for the concept of a highly reactive dication as a potent agent for the cleavage of dihydrogen.

Review


Theoretical insight into the reaction mechanism of the [Fe]-hydrogenase has to date been provided by a small number of studies focusing on small-molecule models for the metal complex at the heart of the active site. Yang and Hall have studied the active site by density functional theory (DFT) methods and considered the differing roles of the metal complex and substrate in dihydrogen cleavage.44,45 The initial mechanism proposed44 was completely reworked45 following the revision of the enzyme crystal structure. In the currently proposed mechanism there are two distinct steps with a “trigger” on coordination by the substrate. The first step in this model is coordination of the dihydrogen to the metal complex, involving a strong hydrogen bond to the hydroxyl group of the pyridone. In the second step, arrival of the substrate triggers cleavage of the dihydrogen with hydride transfer to the substrate (Scheme 15). Yang and Hall account for the lack of observed reactivity in the absence of substrate on the strength of the hydrogen bond when dihydrogen binds to the metal center. They also suggest that activation of dihydrogen takes place via two distinct routes, one involving the cysteine sulfur and the second via a pyridone state. Stiebritz and Reihner have examined the potential analogy between the reaction mechanisms for the [Fe]- and [FeFe]hydrogenases.46 Treating the proximal iron in the [FeFe]hydrogenase as a structural element, they have highlighted

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the similar coordination spheres in the two enzymes (Figure 17). Stiebritz and Reihner interchanged sulfur and nitrogen in the two models to give a number of “swapped” model compounds (Figure 18). DFT calculations showed that these model systems are stable after energy minimization. They postulate that this may indicate a general design principle which is applicable to both classes of hydrogenase.

Figure 17. Analogy between the ligand environment in the [Fe]and [FeFe]-hydrogenases as highlighted by Stiebritz and Reihner.46

Scheme 14. Selective Hydrogenation in Pyridium Salts42,43

Figure 18. “Swapped” ligand models studied by Stiebritz and Reihner.46 Scheme 15. Mechanism Proposed by Yang and Hall Involving Two Possible Activation Pathwaysa,45

a

In the DFT simulation, the substrate and product were approximated by the phenyl-containing molecules shown (10 and 20 , respectively).

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Conclusions

Figure 19. Possible “end-on” intermediate for dihydrogen activation.

Figure 20. Tungsten-hydride complex featuring a W-H 3 3 3 O hydrogen bond.52

Very recently, Dey has reported a DFT study focused on the role of the ligand system in the [Fe]-hydrogenase active site in allowing the metal to bind small molecules.47 The pyridinol-acyl ligand allows delocalization of charge from the metal center on binding anionic ligands such as H-. This makes the iron a potent Lewis acid, unusual for neutral iron(II) centers. Dey concurs with the earlier study by Yang and Hall45 that coordination of dihydrogen is energetically unfavorable in the absence of 1, at least in part due to the need to displace the bound water molecule in the resting state. (42) Corr, M. J.; Gibson, K. F.; Kennedy, A. R.; Murphy, J. A. J. Am. Chem. Soc. 2009, 131, 9174–9175. (43) Corr, M. J.; Roydhouse, M. D.; Gibson, K. F.; Zhou, S.-Z.; Kennedy, A. R.; Murphy, J. A. J. Am. Chem. Soc. 2009, 131, 17980– 17985. (44) Yang, X.; Hall, M. B. J. Am. Chem. Soc. 2008, 130, 14036–14037. (45) Yang, X.; Hall, M. B. J. Am. Chem. Soc. 2009, 131, 10901–10908.

The chemistry of the third hydrogenase is only now coming of age. Understanding of the biological structure of the enzyme has been revealed following the successful purification of large amounts of active enzyme and subsequent crystallization. Advanced synthetic models which mimic the active site of the enzyme are now available, and the next challenge for the bioinorganic chemist is to apply these to understanding the mechanism of the [Fe]-hydrogenase. A key question will be whether the postulated “open” to “closed” transition (Scheme 13) is a required part of the mode of action or whether small-molecule mimics can function in the absence of the enzyme scaffold. Also intriguing is the question of “side-on” versus “end-on” activation of dihydrogen (Figure 19). Notably, the earlier “metal free” mechanism proposed by Berkessel and Thauer38,39 has resonance with the recently developed frustrated Lewis pair mechanism for heterolytic dihydrogen cleavage (or “storage”).48-51 With respect to the [Fe]-hydrogenase this need not be totally dismissed. At both the experimental and theoretical levels a multicentered heterolytic cleavage combining iron 3 3 3 hydrogen and Lewis pair interactions merits exploration. In particular, stabilization of the iron 3 3 3 hydrogen transition state by intramolecular Fe-H 3 3 3 O hydrogen bonding as suggested in Figure 19 might be tenable and has some resonance with known tungsten-hydride W-H 3 3 3 O five-membered cyclic structures (Figure 20).52

Acknowledgment. We thank the University of East Anglia, the EPSRC, and the BBSRC for funding. (46) Stiebritz, M. T.; Reiher, M. Inorg. Chem. 2010, 49, 5818–5823. (47) Dey, A. J. Am. Chem. Soc. 2010, 132, 13892–13901. (48) Welch, G. C.; San Juan, R. R.; Masuda, J. D.; Stephan, D. W. Science 2006, 314, 1124–1126. (49) Stephan, D. W. Org. Biomol. Chem. 2008, 6, 1535–1539. (50) Stephan, D. W. Dalton Trans. 2009, 38, 3129–3136. (51) Stephan, D. W.; Erker, G. Angew. Chem. 2010, 122, 50-81; Angew. Chem., Int. Ed. 2010, 49, 46-76. (52) Fairhurst, S. A.; Henderson, R. A.; Hughes, D. L.; Ibrahim, S. K.; Pickett, C. J. J. Chem. Soc., Chem. Commun. 1995, 32, 1569–1570.

The Third Hydrogenase: More Natural Organometallics

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