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Published in final edited form as: Nat Chem Biol. 2013 Aug 11;9(10):607–609. doi: 10.1038/nchembio.1311

Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic

Julian Esselborn 1, Camilla Lambertz 1,#, Agnieszka Adamska-Venkates 2,#, Trevor Simmons 3,#, Gustav Berggren 3,4, Jens Noth 1, Judith Siebel 2, Anja Hemschemeier 1, Vincent Artero 3, Edward Reijerse 2, Marc Fontecave 3,5, Wolfgang Lubitz 2, Thomas Happe 1,*
PMCID: PMC3795299  EMSID: EMS54738  PMID: 23934246

Abstract

Hydrogenases catalyze the formation of hydrogen. The cofactor (H-cluster) of [FeFe]-hydrogenases consists of a [4Fe-4S]-cluster bridged to a unique [2Fe]-subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe]-subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing novel artificial H2-producing catalysts.

Research on hydrogenase enzymes has gained considerable interest as these biocatalysts efficiently produce molecular hydrogen (H2). In order to develop inexpensive but highly active chemical mimics, detailed knowledge about the structure, reaction mechanism and assembly of the active site in these enzymes is required. Hydrogenases of the [FeFe]-type (HYDA) are the most active H2 producers1. They contain a complex cofactor, the “H-cluster”, consisting of a simple cubane [4Fe-4S]-cluster bound via cysteine to a unique [2Fe]-subcluster in which two [Fe]-atoms are coordinated by CO, CN- and a dithiolate.2,3 (Fig. 1). In the living cell, biosynthesis of the [2Fe]-subcluster requires only three hydrogenase-specific maturases, HYDE, HYDF and HYDG4. HYDF is the scaffold for [2Fe]-subcluster assembly and transfers the cluster to the HYDA protein containing a pre-assembled [4Fe-4S]-cluster (apoHYDA)2,5,6. Numerous model compounds have been synthesized to investigate different features of the [2Fe]-subcluster7. All of the mimics tested so far, including the most structurally relevant ones8,9, have only low H2 evolving efficiencies in vitro. A major challenge is therefore to understand the role of the protein environment in allowing the inorganic clusters to come to full activity10. Notably, a synthetic mimic of the [2Fe]-subcluster can be loaded onto HYDF from Thermotoga maritima in vitro11. This artificially loaded HYDF is able to transfer the di-iron analogues to the apo-form of [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii (apoHYDA1), resulting in a fully active hydrogenase11.

Figure 1. The H-cluster is assembled from a regular [4Fe-4S]-cluster and a unique [2Fe]-subcluster.

Figure 1

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The synthetic [2Fe]-subcluster ([2Fe]MIM) differs from the binuclear subsite of the H-cluster by an additional CO-group. Models of the H- and the [4Fe-4S]-clusters were generated in PyMOL on the basis of PDB structures 3C8Y and 3LX4, respectively. [2Fe]MIM was also modeled in PyMOL according to information given in 11. Some amino acids were deleted to provide an unobstructed view of the clusters. The metal clusters are in the ball-and-stick representation, the coordinating cysteines are shown as sticks. Orange: iron, yellow: sulfur, gray: carbon, blue: nitrogen, red: oxygen.

Little is known about the mechanistic details of the transfer of the [2Fe]-subcluster from HYDF to apoHYDA. However, a function of HYDF in inserting the [2Fe]-subcluster in addition to serving as the scaffold for cluster assembly has been presumed to date. We tested if a [2Fe]-subsite mimic containing an azadithiolate bridge ([2Fe]MIM; Fig. 1)9,11 would be able to integrate into the HYDA1 apo-protein in the absence of HYDF. We combined inactive apoHYDA1 heterologously produced in Escherichia coli with pure [2Fe]MIM solutions. Remarkably, this procedure resulted in high specific hydrogenase activities (Fig. 2a) despite the absence of helper-proteins like HYDF. Both the resulting activities and the molar excess of [2Fe]MIM required to achieve these were similar to or even better than those obtained using [2Fe]MIM-loaded HYDF (HYDFMIM)11 (Fig. 2a). A 2.5-fold excess of [2Fe]MIM over apoHYDA1 sufficed to yield a specific activity as high as HYDA1 maturated in Clostridium acetobutylicum, an established host for heterologous production of active [FeFe]-hydrogenases10 (Fig. 2b). This indicates that the [2Fe]MIM compound was able to autonomously integrate into apoHYDA1 very efficiently.

Figure 2. Various types of HYDA enzymes can be spontaneously activated by [2Fe]MIM and behave like the natural enzymes.

Figure 2

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a, Specific H2 evolution activities of C. reinhardtii HYDA1 after combination of apoHYDA1 with [2Fe]MIM alone or [2Fe]MIM-loaded HYDF. Molar ratios are indicated on the x-axis. b, HYDA1MIM reached the same specific activity as HYDA1 heterologously produced in C. acetobutylicum10 while neither [2Fe]MIM nor apoHYDA1 showed any activities. [2Fe]MIM also activated apoHYDA enzymes of M. elsdenii (HYDA Me MIM) and C. pasteurianum (CpI MIM). We measured H2 evolution using reduced methyl viologen as electron donor. c, H2 production was also achieved in reconstitution assays including pyruvate, acetylCoA, pyruvate:ferredoxin oxidoreductase PFR1 and ferredoxin PETF in the dark17 (pyruvate-driven) or with photosystem 1 (PS1), PETF and the necessary PS1 reducing agents16 in the light (light-driven). a – c, All values shown are mean values from at least four independent experiments. Error bars indicate the standard deviation.

In contrast to C. reinhardtii HYDA1, which consists only of the H-cluster containing H-domain, most bacterial HYDA proteins possess further domains holding additional [FeS]-clusters3,12. The spontaneous integration of [2Fe]MIM into algal apoHYDA1 might therefore be restricted to these single-domain proteins and not suited for broad applications. The crystal structure of C. reinhardtii apoHYDA1 revealed a positively charged channel connecting the protein surface to the active site niche5. As this channel is not observed in holoHYDA enzymes3,13 it might serve as the entry pathway for the [2Fe]-subcluster, closing upon its attachment to the [4Fe-4S]-site. In multi-domain HYDA enzymes the entry-site of the [2Fe]-compound might be obstructed, making a chaperone or maturase indispensable for integrating the [2Fe]-subcluster. Therefore we examined the activity of enzymes from Megasphaera elsdenii (HYDAMe) and C. pasteurianum (CpI) after treatment with [2Fe]MIM. These have two and four additional [FeS]-clusters, respectively, and accordingly longer N-termini3,14. In both cases the apo-enzymes produced in E. coli did not show any H2 evolution activity. However, after in vitro maturation with [2Fe]MIM, specific activities of 308±40 and 2,037±616 μmol H2 · mg HYDAMIM-1· min−1 were achieved by HYDAMeMIM and CpIMIM, respectively (Fig. 2b). These activities were in a similar range as those of native M. elsdenii HYDA15 and heterologously produced CpI10.

We also tested if HYDA1MIM would be able to interact with its natural electron delivery systems. The cellular electron donor of HYDA1 is ferredoxin PETF, and in illuminated algae, the major electron source for H2 production is photosystem 1 (PS1)16. In the dark, pyruvate oxidation and subsequent PETF reduction by pyruvate:ferredoxin oxidoreductase (PFR1) are responsible for H2 generation in Chlamydomonas17. Using in vitro assays in which either PS1 or PFR1 were used as PETF reducing components, H2 evolution activities of HYDA1MIM and HYDA1 were the same (Fig. 2c). The comparable rate of electron transfer between PETF and HYDA1 or HYDA1MIM suggests very similar structural properties and indicates that the in vivo functionality of HYDA1MIM is identical to native HYDA1.

Electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy give detailed information about the electronic structure of the H-cluster and the state of the CO and CN− ligands, respectively18. Therefore, we examined the reconstituted and subsequently purified HYDA1MIM protein by both methods (Fig. 3). In order to obtain well-defined EPR and FTIR spectra of the H-cluster, we treated HYDA1MIM with CO to generate the characteristic Hox-CO state. Indeed, the EPR spectrum of this preparation (Fig. 3a) unequivocally identified it as the native Hox-CO state19. Also its FTIR spectrum (Fig. 3b) showed the CO and CN− bands at the positions previously observed for the native Hox-CO state20. For comparison, Fig. 3 also shows the EPR and FTIR spectra of the starting materials, i.e. apoHYDA1 (Fig. 3a) and [2Fe]MIM (Fig. 3b). EPR spectra of apoHYDA1 indicated typical g-values for [4Fe-4S]-clusters and were very similar to the values observed for apoHYDA1 previously6 (Fig. 3a). The shift of the FTIR bands when going from free [2Fe]MIM to HYDA1MIM-CO (Fig 3b) is consistent with the change of the ligand geometry occurring upon integration of [2Fe]MIM into the polypeptide, which involves its attachment to the [4Fe-4S]-cluster and rearrangement of one CO ligand into a bridging Fe-CO-Fe position (see Fig. 1). In the as-isolated form, [FeFe]-hydrogenases usually show a mixture of redox states, which represent the different electronic configurations of individual steps of the catalytic cycle19. In EPR (Fig. 3a) and FTIR (Fig. 3b) spectra of untreated HYDA1MIM, these signals were also observed. Again, peak shapes and g-tensor values corresponded well to previously published data19,20,21. The spectroscopic data therefore show that the spontaneous integration of [2Fe]MIM into the apoHYDA1 protein results in an H-cluster virtually identical to the native one.

Figure 3. The chemically reconstituted H-cluster is virtually indistinguishable from the native form.

Figure 3

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a, EPR-spectra of apoHYDA1,HYDA1MIM in the CO-inhibited Hox-CO state and as isolated HYDA1MIM comprising all EPR active states. b, FTIR spectra of [2Fe]MIM in solution,of CO-treated HYDA1MIM and as isolated HYDA1MIM . EPR simulations for each state are shown19,21 in the supplementary results (Supplementary Fig. 2a). The g-values are indicated above the spectra. The assignments of the FTIR signals to the different redox states are collected in Supplementary Fig. 2b. All spectra were recorded on two independently prepared samples. One representative result is shown.

One of the four CO ligands of [2Fe]MIM must dissociate to yield active HYDA1MIM (Fig. 1). We verified the release of CO spectroscopically by monitoring the binding of free CO to deoxyhemoglobin over time. This hemoglobin-based assay can sensitively detect dissolved CO22. Indeed, the typical CO-induced shift of the hemoglobin Soret band occurred when apoHYDA1 and [2Fe]MIM were mixed (Supplementary Results, Supplementary Fig. 1a) and the reaction was complete after 2-3 minutes. The release of CO depended linearly on the apoHYDA1 concentration (Supplementary Fig. 1b). We estimated that approximately 0.6 moles of CO were released per mol apoHYDA1.

To test if the spontaneous integration of the [2Fe]-subcluster reported here was strictly specific for the native cofactor [2Fe]MIM, carrying a secondary amine as the dithiolate bridgehead, we attempted to integrate a similar mimic into apoHYDA1. [2Fe]pdt features a 1,3-propanedithiolate and thus differs from [2Fe]MIM only by the head atom of the bridging dithiolate, which is a carbon instead of a nitrogen in this case. [2Fe]pdt, transferred to apoHYDA1 by HYDF, does not form an active H-cluster11. After treating apoHYDA1 with [2Fe]pdt in the absence of HYDF, we could measure no hydrogenase activity accordingly. However, the FTIR spectrum of HYDA1pdt proved the presence of [2Fe]pdt within the protein (Supplementary Fig. 3). In contrast to HYDA1MIM which shows a mixture of states (Fig. 3b) HYDA1pdt is stabilized as a single species resembling the native Hox-state (compare Supplementary Fig. 2b and Supplementary Fig. 3)11. The unassisted integration of [2Fe]pdt shows that the [2Fe]-subcluster integration is not strictly selective. This property could open new avenues to investigate other synthetic cluster derivatives in a protein environment.

In conclusion, the results presented here show that, under in vitro conditions, no [2Fe]-subcluster transferase is needed for HYDA activation and, therefore, offer novel insights into the in vivo maturation process of [FeFe]-hydrogenases. They indicate that after synthesis of the [2Fe]-subsite precursor and its transfer to apoHYDA the last three steps of HYDA activation might also naturally occur without further assistance of maturases. First, the [2Fe]-subcluster must find its way from the protein surface to the pre-assembled [4Fe-4S]-cluster. It was suggested that the water-filled channel reaching from the protein surface to the [4Fe-4S]-cluster in apoHYDA15 might allow an entropically driven insertion of the [2Fe]-subcluster23. Charged residues lining the channel surface are suitable candidates for interacting with the [2Fe]-subsite and directing its steric orientation by providing the correct electrostatic attracting and repelling forces23. Once the [2Fe]-subcluster is positioned at the end of the channel, coordination via a bridging residue to the [4F-e4S]-cluster is required. The coordination of a thioether ligand onto [2Fe] mimics can be oxidatively induced, yielding an intermediate species with a structure closely related to the Hox-CO state24. Finally, the fourth CO-ligand present in [2Fe]MIM, and probably also in the naturally synthesized [2Fe]-subcluster present on HYDF6, dissociates. At which step of H-cluster assembly this happens remains to be elucidated. A transient formation of the Hox-CO state, followed by the release of the excess CO-ligand might be suggested.

Biochemists have long been fascinated by the structure of the [2Fe]-subcluster which, apart from the cysteinyl bridge, has only non-covalent interactions with the protein. One cannot resist the idea that the [2Fe]-subsite may originate from an inorganic complex that spontaneously formed in the pre-biotic world as suggested earlier12. Both HYDF-assisted11 and the here reported unassisted integration of [2Fe]MIM and [2Fe]pdt into apoHYDA1 are powerful demonstrations of the synergy between chemistry and biology. Given the enormous efforts made over the years in both biomimetic chemistry of the [2Fe]-subcluster7 and the elucidation of the functional aspects of the HYDA protein matrix25, bridging the gap between the two fields promises new insights into the chemistry of [FeFe]-hydrogenases. Moreover, the HYDF-independent procedure described here opens up novel areas for hydrogenase research. To date, only a subset of HYDA enzymes has been analyzed in biochemical and biophysical detail, due to the difficulty of generating large amounts of fully active enzymes. The ability to reconstitute apo-hydrogenases even in the absence of maturation factors now permits the analysis of the whole spectrum of HYDA sequences.

In particular the chaperone-independent assembly of (synthetic) clusters will allow us to unravel protein-based structure-function relationships from an entirely new perspective. To date, these are investigated by site-directed mutagenesis of the protein. But with a system of spontaneous cluster assembly at hand, attempts can be made to engineer and test artificial protein environments. This might result in entirely artificial hydrogenases with applications in bio-hydrogen production and bio-fuel cell technology.

Online methods

Heterologous expression and purification of proteins

ApoHYDA1 and apoCpI were produced anaerobically in Escherichia coli BL21(DE3) ΔiscR26 making use of optimized sequences and the pET expression system as described earlier27, but without co-expression of maturase genes. M. elsdenii HYDA was expressed accordingly after cloning of HYDAMe from pT7HME14 into pTSH_hydA1Cr_STII28. Purification of hydrogenases was carried out under strictly anaerobic conditions using a one-step strep-tactin affinity chromatography protocol as described earlier with minor modifications28. Heterologous synthesis of active HYDA1 in Clostridium acetobutylicum ATCC8241428, isolation of PSI29 and plastocyanin from C. reinhardtii30 as well as heterologous expression of PETF16 and PFR117 in E. coli were described earlier.

Preparation of [2Fe]MIM and [2Fe]pdt

Synthesis of [2Fe]MIM and [2Fe]pdt 31,32 followed the previously published protocol9, in the case of [2Fe]pdt with modifications as described earlier11. Crystalline compounds were dissolved in 20 mM HEPES pH 7.5, 100 mM KCl, handled strictly anaerobically and stored at −80°C.

In vitro maturation of [FeFe]-hydrogenases

In vitro maturation of hydrogenases was achieved by incubating 800 ng apo-protein (0.04 μM HYDA1) under strictly anaerobic conditions in 400 μl of 0.1 M potassium phosphate buffer, pH 6.8, with 2 mM sodium dithionite (NaDT) at 25°C for 30 min with a 10-fold molar excess of [2Fe]MIM if not stated otherwise. Subsequent in vitro activity measurements using NaDT-reduced methyl viologen as artificial electron donor were done as previously described33. Maturation of HYDA1MIM for EPR and FTIR measurements was carried out with 150 μM apoHYDA1. The protein was subsequently purified and re-buffered to 0.01 M Tris-HCl, pH 8.0, 2 mM NaDT, by size exclusion chromatography using a NAP™ 5 column (GE Healthcare) and concentrated to 500 μM for EPR and FTIR measurements using Amicon Ultra centrifugal filters 10K (Millipore). Spectra of 12 mM [2Fe]MIM were recorded in 20 mM HEPES buffer pH 7.5, 100 mM KCl.

Pyruvate driven and light driven hydrogen evolution assays

Each pyruvate-driven reaction contained PFR1, PETF and C. reinhardtii HYDA1 or HYDA1MIM, pyruvate and acetylCoA in potassium phosphate buffer, pH 6.8, as described earlier17. The reaction mixtures were incubated for 30 min at 37°C before analyzing the amount of H2 in the gas phase. For light-driven measurements, PSI and plastocyanin from C. reinhardtii, ascorbate and dichlorophenolindophenol were combined with HYDA1 or HYDA1MIM and PETF as previously published16. These reaction mixtures were illuminated for 30 min at 30°C using monochromatic white and red light (250 μEinstein).

Detection of CO by a hemoglobin-based assay

Bovine hemoglobin (Sigma-Aldrich) was reduced to deoxyhemoglobin (Hb) under strictly anaerobic conditions with NaDT and was brought to ~ 4.5 μM hemoglobin in 100 mM potassium phosphate buffer pH 6.8 with 2 mM NaDT. Spectra and single wavelength kinetics were taken with a UV-2450 spectrophotometer (Shimadzu) at room temperature in sealed 1 ml micro UV-cuvettes to keep anaerobic conditions. [2Fe]MIM was added with gastight syringes (Hamilton) from sealed, anaerobic vials to 10 times the intended concentration of apoHYDA1 (2 μM if not stated otherwise). After each injection thorough mixing was ensured by inverting the cuvette several times. During the whole process the absorbance at 419 nm characteristic for the maximum of the Soret peak of carbonmonoxyhemoglobin (HbCO) was recorded. Concentrations of HbCO were calculated using the difference of the molar absorption coefficients of Hb and HbCO at 419 nm, which can be calculated from spectra of known concentrations of fully reduced Hb and fully saturated HbCO, respectively. The heme concentration could accurately be measured using published molar absorption coefficients of characteristic bands in the visible range34.

EPR and FTIR analyses

FTIR spectra were obtained with a Bruker IFS 66v/s FTIR spectrometer equipped with a Bruker MCT (mercury cadmium telluride) detector. The spectra were accumulated in the double-sided, forward-backward mode with 1000 scans and a resolution of 2 cm−1 at 15°C. Data processing was facilitated by home written routines in the MATLAB program environment.

Q-band EPR spectra were measured using the 2 pulse echo-detected EPR technique. The length of the π/2 and π MW pulses were set to 16 ns and 32 ns respectively, separated with τ = 500ns. The shot repetition time was set to 1 ms. All measurements were performed on a home build Q-band pulse spectrometer. The temperature was controlled in a custom-built cryofree cryostat from Advanced Research Systems (base temperature 8 K). The sample was accommodated in a homebuilt Q-band ENDOR resonator insert35.

Statistical analyses

All hydrogenase activity tests were done at least four times. Values shown are averages, error bars show the standard deviations. EPR and FTIR analyses were done on two independently prepared samples.

Supplementary Material

SI

Acknowledgments

This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) (Bio-H2) (to T.H. and W.L.), the Max Planck Society, the French National Research Agency (ANR, NiFe–Cat ANR–10–BLAN–711 and Labex program ARCANE, 11– LABX–003) (to T.S.,V.A and M.F) and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013/ERC Grant Agreement n.306398) (to V.A.). GB gratefully acknowledges Bengt Lundqvist minnesfond, FORMAS (contract number 213-2010-563) and the Swedish royal academy of sciences. T.H. gratefully acknowledges support from the Deutsche Forschungsgemeinschaft (HA 255/2-1) and the Volkswagen foundation (LigH2t). J.E. is financed by the Studienstiftung des deutschen Volkes.

Footnotes

Competing Financial Interests The authors declare no competing financial interests.

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