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Structural and functional characterization of the hydrogenase-maturation HydF protein

Abstract

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H+ and H2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN and azapropanedithiolate (adt2−) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe2(adt)(CO)4(CN)2]2− is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.

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Figure 1: Structures of the H cluster, chemical analogs of the 2Fe subcluster, and the 6Fe units in HydF hybrids described in this study.
Figure 2: EPR and structural characterization of FeS–HydF from T. melanesiensis.
Figure 3: Hyperfine sublevel correlation (HYSCORE) spectra of reduced FeS–TmeHydF.
Figure 4: FTIR spectra of 1–TmeHydF and 2–TmeHydF.
Figure 5: Hydrogen evolution activity of 2–HydF and 2–HydA.

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Acknowledgements

Diffraction data were collected at the synchrotron SOLEIL, beamlines Proxima 1 and Proxima 2 (Saint-Aubin, France). We are most grateful to the beamline groups for making these experiments possible. V.A., M.A. and S.R. acknowledge support from the French National Research Agency (Labex program ARCANE, ANR-11-LABX-0003-01). G.C., L.P., C.P. and M.F. acknowledge support from the French National Research Agency (Labex program DYNAMO, ANR-11-LABX-0011) and from Fondation de l'Orangerie for Individual Philanthropy and its donors.

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Contributions

G.C., L.P. and M.F. designed the study; G.C. performed protein purification, characterization and crystallogenesis, and sample preparation for spectroscopy experiments; C.P. performed mutagenesis and characterization of mutant proteins; G.C. and C.P. assayed hydrogenase activity; L.P. determined the three-dimensional structures; A.A.-V., E.R. and W.L. performed and analyzed the EPR, HYSCORE and FTIR experiments; V.A. and S.R. contributed to synthetic chemistry; M.A. and L.P. contributed to molecular biology; and M.F., G.C., L.P. and E.R. wrote the manuscript with input from all other authors.

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Correspondence to Marc Fontecave.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–4, Supplementary Figures 1–20 and Supplementary Note 1. (PDF 3746 kb)

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Caserta, G., Pecqueur, L., Adamska-Venkatesh, A. et al. Structural and functional characterization of the hydrogenase-maturation HydF protein. Nat Chem Biol 13, 779–784 (2017). https://doi.org/10.1038/nchembio.2385

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