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Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase - Nature

  • ️Lubitz, Wolfgang
  • ️Mon Jan 26 2015

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Crystallographic data and coordinates have been deposited in the Protein Data Bank under accession numbers 4U9H (Ni-RUH) and 4U9I (Ni-RH), respectively.

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Acknowledgements

We thank P. Malkowski for her help with the sample preparation and K. Krause for critically reading the manuscript. We are grateful to the staff of BESSYII MX-beamline (Helmholtz-Zentrum Berlin, Germany) for their help during the data collection. The work was supported by the Max Planck Society, the Bundesministerium für Bildung und Forschung (BMBF) (03SF0355C), EU/Energy Network project SOLAR-H2 (FP7 contract 212508), and in part by the Cluster of Excellence RESOLV (EXC1069) funded by the Deutsche Forschungsgemeinschaft.

Author information

Author notes

  1. Koji Nishikawa

    Present address: Present address: Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.,

Authors and Affiliations

  1. Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, Germany,

    Hideaki Ogata, Koji Nishikawa & Wolfgang Lubitz

Authors

  1. Hideaki Ogata

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  2. Koji Nishikawa

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  3. Wolfgang Lubitz

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Contributions

K.N. performed the sample preparation. K.N. and H.O. collected the X-ray diffraction data and performed the data analysis. H.O. measured the FTIR spectra. H.O. and W.L. designed and coordinated the project and wrote the manuscript.

Corresponding authors

Correspondence to Hideaki Ogata or Wolfgang Lubitz.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Electron density maps of the [FeS] clusters and the Mg binding site.

Stereo views of the 2Fo − Fc electron density map contoured at 4σ and the Fo − Fc omit map contoured at 5σ of Ni-RUH data are shown in blue and green, respectively. The green mesh indicates the electron density of the hydrogen atoms. a, Proximal [4Fe–4S] cluster. b, Medial [3Fe–4S] cluster. c, Distal [4Fe–4S] cluster. The superscript S indicates that the coordinated amino-acid residues are in the small subunit. d, The Mg at the carboxy (C) terminus and water molecules involved in the hydrogen bond networks. Stereo view of the 2Fo − Fc electron density map contoured at 4σ and the Fo − Fc omit map contoured at 3σ of Ni-RUH data are shown in blue and green, respectively.

Extended Data Figure 2 FTIR spectroscopy.

FTIR spectra recorded under anoxic conditions with H2 (1.5 ± 0.5%) and N2 (98.5 ± 0.5%) at T = 293 K. Several crystals together with a small amount of the surrounding crystallization buffer in the same crystallization well used for X-ray diffraction experiments were loaded in the infrared cell (CaF2 windows). a, Crystals from Ni-RUH. b, Crystals from Ni-RH. The peaks, νCO (1945 cm−1) and νCNs (2060 and 2074 cm−1), indicate the Ni-R1 state. The peaks, 1930/1931 cm−1 and 1960 cm−1, indicate νCO of the Ni-R2 and Ni-C states, respectively. The peak (at ∼1986 cm−1) marked with an asterisk is probably caused by degraded material in the crystallization buffer. The ratio of Ni-R1, Ni-R2 and Ni-C in Ni-RUH was estimated to 78%, 18% and 4%, respectively. In Ni-RH the ratio of Ni-R1, Ni-R2 and the species giving rise to the peak at 1986 cm−1 was estimated to 85%, 9% and 5%, respectively. The detection limit was about 1–2%.

Extended Data Figure 3 Estimated coordinate errors of the amino-acid residues and position of the hydrogen atoms.

a, The estimated standard deviation of the bond length of the amino-acid residues (carbon, black; nitrogen, blue; oxygen, red) except for the hydrogen atoms and the metal atoms. Atoms with large B-factors (>30 Å2) were omitted. b, The electron density map of Gly 550 (2Fo − Fc map, blue, contoured at 4σ; Fo − Fc omit map, green, contoured at 3.6σ). The green spheres show the peak positions of the residual electron density in the Fo − Fc omit map. The position of the hydrogen atoms by the riding models (that is, expected position of the electron density for X-ray and of the nuclei for neutron diffraction) is shown both for X-ray (labelled Hx) and for neutron diffraction (HN), respectively. For X-ray crystallography it is well known that the electron density of the hydrogen atoms appears closer to the attached heavier atoms17. The peak positions of the residual electron density are shifted compared to the riding models (∼0.1 Å for X-ray and ∼0.2 Å for neutron diffraction).

Extended Data Figure 4 Electron density map of the active site.

Stereo view of the 2Fo − Fc electron density map contoured at 4σ and the Fo − Fc omit map contoured at 5σ of Ni-RUH data are shown in blue and green, respectively. The green mesh indicates the electron density of the hydrogen atoms. The colour code of the ball and stick representation is nickel (green), iron (orange), carbon (grey), nitrogen (blue), oxygen (red), sulphur (yellow) and hydrogen (white). The figure highlights the electron density (the Fo − Fc omit map in green) of the hydrogen atoms around the [NiFe] active site. One hydrogen atom attached to Cβ(Cys 546) was invisible, but the rest of the hydrogen atoms of the four cysteine residues bound to the [NiFe] active site could be clearly identified. The electron density of the hydride bridge is slightly larger than that of the hydrogen atoms of the cysteine residues because of the two electrons of the hydride (see Extended Data Fig. 5).

Extended Data Figure 5 Plots of relative averaged electron density versus temperature factor.

The hydrogen atoms of the selected 50 amino-acid residues that are located within 10 Å from the Ni atom are shown by the black dots. Atoms with large B-factors (>10 Å2) were omitted. The red and blue squares indicate the hydride bridge and the proton attached to the Cys 546, respectively. The ellipsoid represents the 95% limit for a fit of the hydrogen atoms. The relative averaged electron density value of the hydride (6.7) is twofold larger than the centre of the ellipsoids (3.1). The plot clearly shows the larger electron density for the hydride (H−) compared with the other hydrogens and excludes the possibility that this density results from a heavier atom (such as oxygen, for example).

Extended Data Figure 6 Comparison of the reduced and oxidized states of the [NiFe] active site.

The 2Fo − Fc electron density map (contoured at 4σ, blue) and the Fo − Fc omit map (contoured at 6σ, green) shows the data from Ni-RH. The ball and stick models show the superimposed structures of the [NiFe] active site. The structure alignment was done using all amino-acid residues. The reduced state (Ni-RUH and Ni-RH) and the oxidized states (Ni-A: PDB accession numbers 1WUH and 1WUI; Ni-B: PDB accession number 1WUJ)27 are shown in white and red, respectively. The residual electron density (green mesh) at the bridging position indicates that no contamination of the oxidized state in the crystal structure is presented. The bond lengths are listed in the Extended Data Table 2.

Extended Data Figure 7 Geometry of the Ni-R and Ni-C states of [NiFe] hydrogenase.

Bond lengths (in ångströms) and angles (in degrees) for the coordination of the nickel ion at the active site are shown. a, Ni-RUH data. b, Ni-R state from DFT calculations (Ni2+)28. The cysteine residue (Cys 546) is protonated in both X-ray structure and DFT calculations. c, Ni-C state from DFT calculations (Ni3+), here the Cys 546 is not protonated28. The Ni–S(Cys 549) bond length in the Ni-RUH data (2.54 Å) is significantly longer than the other Ni–S bond lengths (∼2.2 Å). These structural features are in good agreement with recent DFT calculations, indicating a near square pyramidal geometry at the Ni2+, indicating a low-spin state28. In the Ni-C state, the four Ni–sulphur bond lengths are almost equal (2.3 ± 0.1 Å), and the angle is ∼167° (S(Cys 84)–Ni–S(Cys 546)).

Extended Data Figure 8 Possible proton transfer pathways.

a, Ball and stick representation of the active [NiFe] site, the amino-acid residues and the water molecules that are related to the possible proton pathways (1–3). The black dotted lines indicate the hydrogen bond networks. Path 2 and Path 3 are less favourable owing to participation of non-conserved amino acids (for example, Glu 57S in Path 2 is replaced by Leu/Ile in case of the [NiFeSe] hydrogenases breaking the hydrogen bond. Leu 57 in Path 3 is replaced by Met in the case of the membrane-bound [NiFe] hydrogenases, which also breaks the hydrogen bond network26). b, Electron density map (2Fo − Fc map, 4σ, blue; Fo − Fc omit map, 2.5σ, green) of part of the proton pathway (Path 1) involving protonated His 36. The green mesh indicates the electron density of the hydrogen atoms. c, Possible proton pathway (Path 3) and the coordination of the Mg site. The electron density is only shown for the water molecules for simplicity. The black dotted lines show Path 3 and the red dotted lines show the coordination of the Mg centre and of its hydrogen bond network. d, The sequence alignment related to the proton transfer pathway for different species. The amino-acid residues marked with stars are shown in Fig. 3a. DvMF, D. vulgaris Miyazaki F; Av, Allochromatium vinosum; Dd, Desulfovibrio desulfuricans; Df, Desulfovibrio fructosovorans; Dg, Desulfovibrio gigas; Ec, Escherichia coli; Hm, Hydrogenovibrio marinus; Re, Ralstonia eutropha; Se, Salmonella enterica; Db, [NiFeSe] hydrogenase from Desulfomicrobium baculatum; DvH, [NiFeSe] hydrogenase from D. vulgaris Hildenborough. The PDB entries are also given. The references describing the structures of these hydrogenases are found in ref. 1.

Extended Data Table 1 Data collection and refinement statistics*

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Extended Data Table 2 Bond lengths of the [NiFe] active site and the Ni-complexes

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Ogata, H., Nishikawa, K. & Lubitz, W. Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase. Nature 520, 571–574 (2015). https://doi.org/10.1038/nature14110

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  • Received: 18 August 2014

  • Accepted: 20 November 2014

  • Published: 26 January 2015

  • Issue Date: 23 April 2015

  • DOI: https://doi.org/10.1038/nature14110