Biomimetic assembly and activation of [FeFe]-hydrogenases


Hydrogenases are the most active molecular catalysts for hydrogen production and uptake1,2, and could therefore facilitate the development of new types of fuel cell3,4,5. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN ligands6,7. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation8. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite1, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases9,10. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

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Figure 1: Structures of the di-iron clusters discussed in the study.
Figure 2: Normalized FTIR spectra recorded in liquid solution at 15 °C.
Figure 3: Continuous wave and pulsed EPR spectra of 1–HydF.
Figure 4: Specific hydrogenase activity of reconstituted HydA1.


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G.B. acknowledges support from the Bengt Lundqvist Minnesfond, FORMAS (contract number 213-2010-563) and the Swedish Royal Academy of Sciences. This work was supported by the French National Research Agency (ANR) through grant 07-BLAN-0298-01 and the Labex programme (ARCANE, 11-LABX-003). V.A. acknowledges support from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013/ERC Grant Agreement no. 306398). T.H. was supported by the Deutsche Forschungsgemeinschaft (HA 255/2-1), the BMBF (Bio-H2) and the Volkswagen foundation (LigH2t). A.A., E.R. and W.L. thank the Max Planck Society for financial support, and A. Silakov for providing the FTIR processing software.

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G.B., V.A., M.A., W.L., T.H. and M.F. designed the research; G.B. and T.R.S. prepared and characterized synthetic complexes and hybrid species; C.L., J.E. and G.B. contributed to maturation experiments and H2 evolution measurements; A.A. and C.L. performed FTIR measurements; G.B. and S.G. performed EPR measurements; J.-M.M. did DFT calculations; and M.F., G.B, E.R. and V.A. wrote the paper.

Correspondence to M. Fontecave.

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Berggren, G., Adamska, A., Lambertz, C. et al. Biomimetic assembly and activation of [FeFe]-hydrogenases. Nature 499, 66–69 (2013).

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