De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy

Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI: http://dx.doi.org/10.7554/eLife.00218.001

[1]  M. Field,et al.  Gas access to the active site of Ni-Fe hydrogenases probed by X-ray crystallography and molecular dynamics , 1997, Nature Structural Biology.

[2]  Z. Zhou,et al.  3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy , 2008, Nature.

[3]  Liam J. McGuffin,et al.  Protein structure prediction servers at University College London , 2005, Nucleic Acids Res..

[4]  X Vernede,et al.  The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center. , 1999, Structure.

[5]  H. D. Peck,et al.  Carboxy‐terminal processing of the large subunit of [NiFe] hydrogenases , 1993, FEBS letters.

[6]  Ivo Atanasov,et al.  Atomic Structure of Human Adenovirus by Cryo-EM Reveals Interactions Among Protein Networks , 2010, Science.

[7]  C. Walsh,et al.  8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies. , 1987, Biochemistry.

[8]  D. Jahn,et al.  Aspartate 141 Is the Fourth Ligand of the Oxygen-sensing [4Fe-4S]2+ Cluster of Bacillus subtilis Transcriptional Regulator Fnr* , 2010, The Journal of Biological Chemistry.

[9]  W. Lubitz,et al.  The crystal structure of the [NiFe] hydrogenase from the photosynthetic bacterium Allochromatium vinosum: characterization of the oxidized enzyme (Ni-A state). , 2010, Journal of molecular biology.

[10]  M. Adams,et al.  1H NMR investigation of the electronic and molecular structure of the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus. Identification of Asp 14 as a cluster ligand in each of the four redox states. , 1995, Biochemistry.

[11]  Ivo Atanasov,et al.  Atomic model of CPV reveals the mechanism used by this single-shelled virus to economically carry out functions conserved in multishelled reoviruses. , 2011, Structure.

[12]  R. Huber,et al.  [NiFe]-Hydrogenase maturation endopeptidase: structure and function. , 2005, Biochemical Society transactions.

[13]  C. Soares,et al.  [NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c3 , 2000, JBIC Journal of Biological Inorganic Chemistry.

[14]  A. Volbeda,et al.  X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli , 2012, Proceedings of the National Academy of Sciences.

[15]  O. Dym,et al.  Sequence‐structure analysis of FAD‐containing proteins , 2001, Protein science : a publication of the Protein Society.

[16]  Dong-Hua Chen,et al.  De novo backbone trace of GroEL from single particle electron cryomicroscopy. , 2008, Structure.

[17]  M. Levitt,et al.  Mechanism of Folding Chamber Closure in a Group II Chaperonin , 2010, Nature.

[18]  Arnim Wiezer,et al.  Complete Genome Sequence of Methanothermobacter marburgensis, a Methanoarchaeon Model Organism , 2010, Journal of bacteriology.

[19]  Jiro Nomata,et al.  X-ray crystal structure of the light-independent protochlorophyllide reductase , 2010, Nature.

[20]  B. Böttcher,et al.  Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy , 1997, Nature.

[21]  N. Yasuoka,et al.  Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis. , 1997, Structure.

[22]  D. McLachlan.  X-ray crystal structure , 1957 .

[23]  Wah Chiu,et al.  4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement , 2010, Proceedings of the National Academy of Sciences.

[24]  F. DiMaio,et al.  Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus , 2011, Proceedings of the National Academy of Sciences.

[25]  M. Hoppert,et al.  Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg , 1994, Journal of bacteriology.

[26]  Martin Grininger,et al.  Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy , 2010, Proceedings of the National Academy of Sciences.

[27]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[28]  W. Chiu,et al.  A 11.5 A single particle reconstruction of GroEL using EMAN. , 2001, Journal of molecular biology.

[29]  C. Walsh,et al.  Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5-deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum delta H. , 1990, Biochemistry.

[30]  C. Walsh,et al.  Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase , 1987, Journal of bacteriology.

[31]  Y. Higuchi,et al.  Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase , 2011, Nature.

[32]  E. Muth,et al.  Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae. , 1987, European journal of biochemistry.

[33]  R. Thauer,et al.  Growth parameters (Ks, μmax, Ys) of Methanobacterium thermoautotrophicum , 1980, Archives of Microbiology.

[34]  C. Walsh,et al.  8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization. , 1987, Biochemistry.

[35]  Nikolaus Grigorieff,et al.  Atomic model of an infectious rotavirus particle , 2010, The EMBO journal.

[36]  C. Spahn,et al.  The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre , 2011, Nature.

[37]  P. Vignais,et al.  Occurrence, classification, and biological function of hydrogenases: an overview. , 2007, Chemical reviews.

[38]  Xing Zhang,et al.  3.3 Å Cryo-EM Structure of a Nonenveloped Virus Reveals a Priming Mechanism for Cell Entry , 2010, Cell.

[39]  R. Conrad,et al.  Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria , 2011, Applied and Environmental Microbiology.

[40]  Michel Frey,et al.  Crystal structure of the nickel–iron hydrogenase from Desulfovibrio gigas , 1995, Nature.

[41]  Eberhard Warkentin,et al.  Structural basis of the hydride transfer mechanism in F(420)-dependent methylenetetrahydromethanopterin dehydrogenase. , 2009, Biochemistry.

[42]  N Grigorieff,et al.  Resolution measurement in structures derived from single particles. , 2000, Acta crystallographica. Section D, Biological crystallography.

[43]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[44]  Anne-Kristin Kaster,et al.  Hydrogenases from methanogenic archaea, nickel, a novel cofactor, and H2 storage. , 2010, Annual review of biochemistry.

[45]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[46]  J. J. Fernández,et al.  Sharpening high resolution information in single particle electron cryomicroscopy. , 2008, Journal of structural biology.

[47]  Nikolaus Grigorieff,et al.  Subunit interactions in bovine papillomavirus , 2010, Proceedings of the National Academy of Sciences.

[48]  S. Harrison,et al.  Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction , 2008, Proceedings of the National Academy of Sciences.

[49]  Wen Jiang,et al.  EMAN2: an extensible image processing suite for electron microscopy. , 2007, Journal of structural biology.

[50]  M. Levitt,et al.  Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure. , 2011, Structure.

[51]  K. M. Shaw,et al.  Properties of the particulate enzyme F420-reducing hydrogenase isolated from Methanospirillum hungatei , 1987 .

[52]  W. Wooster,et al.  Crystal structure of , 2005 .

[53]  S. Shima,et al.  [28] Tetrahydromethanopterin-specific enzymes from Methanopyrus kandleri , 2001 .

[54]  Robert M Glaeser,et al.  Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM. , 2011, Journal of structural biology.

[55]  M. Levitt,et al.  Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle , 2011, The EMBO journal.

[56]  S. Shima,et al.  Tetrahydromethanopterin-specific enzymes from Methanopyrus kandleri. , 2001, Methods in enzymology.

[57]  Eric F. Johnson,et al.  A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii* , 2005, Journal of Biological Chemistry.

[58]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[59]  W. Chiu,et al.  Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy. , 2004, Structure.

[60]  Shaoxia Chen,et al.  Prevention of overfitting in cryo-EM structure determination , 2012, Nature Methods.

[61]  P. Matias,et al.  The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, "as-isolated" state. , 2010, Journal of molecular biology.