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Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG

Nature volume 465, pages 248–251 (2010)Cite this article

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Abstract

Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism1,2. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases3,4 (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands5,6. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear7,8,9,10. Here we show the structure of HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery11,12 and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery7,8,9,10. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

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Figure 1: Ball-and-stick representation of the H-cluster in [FeFe]-hydrogenase from Clostridium pasteurianum29.
Figure 2: X-ray crystal structure of C. reinhardtii HydA ΔEFG determined to a resolution of 1.97 Å, compared with HydA from CpI.
Figure 3: Active-site comparison between C. reinhardtii HydA ΔEFG and HydA from CpI.
Figure 4: Channels for insertion into hydrogenase and nitrogenase during complex Fe–S-cluster assembly.

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Protein Data Bank

Data deposits

Coordinates and structure factors of C. reinhardtii HydAΔEFG have been deposited in the Protein Data Bank under the accession code 3LX4.

References

  1. Drennan, C. L. & Peters, J. W. Surprising cofactors in metalloenzymes. Curr. Opin. Struct. Biol. 13, 220–226 (2003)

    Article  CAS  Google Scholar 

  2. Fontecilla-Camps, J. C., Amara, P., Cavazza, C., Nicolet, Y. & Volbeda, A. Structure-function relationships of anaerobic gas-processing metalloenzymes. Nature 460, 814–822 (2009)

    Article  ADS  CAS  Google Scholar 

  3. Vignais, P. M. & Billoud, B. Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 107, 4206–4272 (2007)

    Article  CAS  Google Scholar 

  4. Shima, S. & Thauer, R. K. A third type of hydrogenase catalyzing H2 activation. Chem. Rec. 7, 37–46 (2007)

    Article  CAS  Google Scholar 

  5. Peters, J. W., Lanzilotta, W. N., Lemon, B. J. & Seefeldt, L. C. X-ray crystal structure of the Fe-only hydrogenase (Cpl) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853–1858 (1998)

    Article  ADS  CAS  Google Scholar 

  6. Nicolet, Y., Piras, C., Legrand, P., Hatchikian, C. E. & Fontecilla-Camps, J. C. Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Structure 7, 13–23 (1999)

    Article  CAS  Google Scholar 

  7. Nicolet, Y. et al. X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima . J. Biol. Chem. 283, 18861–18872 (2008)

    Article  CAS  Google Scholar 

  8. McGlynn, S. E. et al. HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis. FEBS Lett. 582, 2183–2187 (2008)

    Article  CAS  Google Scholar 

  9. Pilet, E. et al. The role of the maturase HydG in [FeFe]-hydrogenase active site synthesis and assembly. FEBS Lett. 583, 506–511 (2009)

    Article  CAS  Google Scholar 

  10. Mulder, D. W. et al. Activation of HydAΔEFG requires a preformed [4Fe-4S] cluster. Biochemistry 48, 6240–6248 (2009)

    Article  CAS  Google Scholar 

  11. Lill, R. Function and biogenesis of iron-sulphur proteins. Nature 460, 831–838 (2009)

    Article  ADS  CAS  Google Scholar 

  12. Johnson, D. C., Dean, D. R., Smith, A. D. & Johnson, M. K. Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281 (2005)

    Article  CAS  Google Scholar 

  13. Schwarz, G., Mendel, R. R. & Ribbe, M. W. Molybdenum cofactors, enzymes and pathways. Nature 460, 839–847 (2009)

    Article  ADS  CAS  Google Scholar 

  14. Posewitz, M. C. et al. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. J. Biol. Chem. 279, 25711–25720 (2004)

    Article  CAS  Google Scholar 

  15. Rubach, J. K., Brazzolotto, X., Gaillard, J. & Fontecave, M. Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima . FEBS Lett. 579, 5055–5060 (2005)

    Article  CAS  Google Scholar 

  16. Brazzolotto, X. et al. The [Fe-Fe]-hydrogenase maturation protein HydF from Thermotoga maritima is a GTPase with an iron-sulfur cluster. J. Biol. Chem. 281, 769–774 (2006)

    Article  CAS  Google Scholar 

  17. McGlynn, S. E., Mulder, D. W., Shepard, E. M., Broderick, J. B. & Peters, J. W. Hydrogenase cluster biosynthesis: organometallic chemistry nature’s way. Dalton Trans. 22, 4274–4285 (2009)

    Article  Google Scholar 

  18. Peters, J. W., Szilagyi, R. K., Naumov, A. & Douglas, T. A radical solution for the biosynthesis of the H-cluster of hydrogenase. FEBS Lett. 580, 363–367 (2006)

    Article  CAS  Google Scholar 

  19. Balk, J., Pierik, A. J., Netz, D. J., Muhlenhoff, U. & Lill, R. The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins. EMBO J. 23, 2105–2115 (2004)

    Article  CAS  Google Scholar 

  20. Peters, J. W. et al. Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36, 1181–1187 (1997)

    Article  CAS  Google Scholar 

  21. Einsle, O. et al. Nitrogenase MoFe-protein at 1.16 Å resolution: a central ligand in the FeMo-cofactor. Science 297, 1696–1700 (2002)

    Article  ADS  CAS  Google Scholar 

  22. Schmid, B. et al. Structure of a cofactor-deficient nitrogenase MoFe protein. Science 296, 352–356 (2002)

    Article  ADS  CAS  Google Scholar 

  23. Fani, R., Gallo, R. & Lio, P. Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes. J. Mol. Evol. 51, 1–11 (2000)

    Article  ADS  CAS  Google Scholar 

  24. Meyer, J. [FeFe] hydrogenases and their evolution: a genomic perspective. Cell. Mol. Life Sci. 64, 1063–1084 (2007)

    Article  CAS  Google Scholar 

  25. Hug, L. A., Stechmann, A. & Roger, A. J. Phylogenetic distributions and histories of proteins involved in anaerobic pyruvate metabolism in eukaryotes. Mol. Biol. Evol. 27, 311–324 (2010)

    Article  CAS  Google Scholar 

  26. Rubio, L. M. & Ludden, P. W. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 62, 93–111 (2008)

    Article  CAS  Google Scholar 

  27. Georgiadis, M. M. et al. Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii . Science 257, 1653–1659 (1992)

    Article  ADS  CAS  Google Scholar 

  28. DeLano, W. L. PyMOL Molecular Viewer 〈http://www.pymol.org〉 (2002)

    Google Scholar 

  29. Pandey, A. S., Harris, T. V., Giles, L. J., Peters, J. W. & Szilagyi, R. K. Dithiomethylether as a ligand in the hydrogenase H-cluster. J. Am. Chem. Soc. 130, 4533–4540 (2008)

    Article  CAS  Google Scholar 

  30. Silakov, A., Wenk, B., Reijerse, E. & Lubitz, W. 14N HYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge. Phys. Chem. Chem. Phys. 11, 6592–6599 (2009)

    Article  CAS  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  32. Collaborative. Computation Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  33. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  34. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999)

    Article  CAS  Google Scholar 

  35. Terwilliger, T. C. SOLVE and RESOLVE: automated structure solution and density modification. Methods Enzymol. 374, 22–37 (2003)

    Article  CAS  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  37. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    Article  ADS  Google Scholar 

  38. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  39. Ten Eyck, L. F. Crystallographic fast Fourier transforms. Acta Crystallogr. A 29, 183–191 (1973)

    Article  ADS  CAS  Google Scholar 

  40. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    Article  CAS  Google Scholar 

  41. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003)

    Article  Google Scholar 

  42. Le, S. Q. & Gascuel, O. An improved general amino acid replacement matrix. Mol. Biol. Evol. 25, 1307–1320 (2008)

    Article  CAS  Google Scholar 

  43. Abascal, F., Zardoya, R. & Posada, D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105 (2005)

    Article  CAS  Google Scholar 

  44. Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003)

    Article  CAS  Google Scholar 

  45. Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755 (2001)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a US Air Force Office of Scientific Research Multidisciplinary University Research Initiative Award (FA9550-05-01-0365, J.W.P.) and the NASA Astrobiology Institute (NAI)-funded Astrobiology Biogeocatalysis Research Center (NNA08C-N85A, J.B.B. and J.W.P.). E.S.B. was supported by a NAI postdoctoral fellowship. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology programme is supported by the US Department of Energy, Office of Biological and Environmental Research, the US National Institutes of Health, National Center for Research Resources, Biomedical Technology programme, and the US National Institute of General Medical Sciences.

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Authors and Affiliations

  1. Astrobiology Biogeocatalysis Research Center,,

    David W. Mulder, Eric S. Boyd, Ranjana Sarma, Rachel K. Lange, James A. Endrizzi, Joan B. Broderick & John W. Peters

  2. Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, USA,

    David W. Mulder, Eric S. Boyd, Ranjana Sarma, Rachel K. Lange, James A. Endrizzi, Joan B. Broderick & John W. Peters

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Contributions

Author Contributions The structural work was conducted by D.W.M. with contributions from R.S. and J.A.E. E.S.B. led the phylogenetic work with contributions from R.K.L. J.W.P. supervised the work with assistance from J.B.B. D.W.M., E.S.B. and J.W.P. led the manuscript preparation with contributions from J.B.B., R.S., R.K.L. and J.A.E.

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Correspondence to John W. Peters.

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Mulder, D., Boyd, E., Sarma, R. et al. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG. Nature 465, 248–251 (2010). https://doi.org/10.1038/nature08993

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