Archives of Microbiology

, Volume 151, Issue 4, pp 307–313 | Cite as

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

  • S. F. Baron
  • D. S. Williams
  • H. D. May
  • P. S. Patel
  • H. C. Aldrich
  • J. G. Ferry
Original Papers


The ultrastructural locations of the coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F420-dependent formate dehydrogenase activity or F420-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.

Key words

Methanobacterium formicicum Formate dehydrogenase F420-hydrogenase Immunogold Ultrastructure Methanogen 



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  1. Aldrich HC, Beinborm DB, Bokranz M, Schönheit P (1987a) Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium formicicum. Arch Microbiol 147: 190–194Google Scholar
  2. Aldrich HC, Beimborn DB, Schönheit P (1987b) Creation of artifactual internal membranes during fixation of Methanobacterium thermoautotrophicum. Can J Microbiol 33: 844–849Google Scholar
  3. Armbruster BL, Kellenberger E (1986) Low temperature embedding. In: Aldrich HC, Todd WJ (eds) Ultrastructure techniques for microorganisms. Plenum Press, New York London, pp 267–295Google Scholar
  4. Barber MJ, Siegel LM, Schauer NL, May HD, Ferry JG (1983) Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers. J Biol Chem 258: 10839–10845PubMedGoogle Scholar
  5. Barber MJ, May HD, Ferry JG (1986) Inactivation of formate dehydrogenase from Methanobacterium formicicum by cyanide. Biochemistry 25: 8150–8155Google Scholar
  6. Baron SF, Brown DP, Ferry JG (1987) Locations of the hydrogenase of Methanobacterium formicicum after subcellular fractionation of cell extract. J Bacteriol 169: 3823–3825PubMedGoogle Scholar
  7. Bell GR, LeGall J, Peck HD (1974) Evidence for the periplasmic location of hydrogenase in Desulfovibrio gigas. J Bacteriol 120: 994–997PubMedGoogle Scholar
  8. Bendayan M (1984) Enzyme-gold electron microscopic cytochemistry, a new affinity approach for the ultrastructural localization of macromolecules. J Electr Micr Tech 1: 349–372Google Scholar
  9. Blaut M, Gottschalk G (1985) Evidence for a chemiosmotic mechanism of ATP synthesis in methanogenic bacteria. TIBS 10: 486–489Google Scholar
  10. Bradford M (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254CrossRefPubMedGoogle Scholar
  11. Fox JA, Livingston DJ, Orme-Johnson WH, Walsh CT (1987) 8-Hydroxy-5-deazaflavin-reducig hydrogenase from Methanobacterium thermoautotrophicum. 1. Purification and characterization. Biochemistry 26: 4219–4227PubMedGoogle Scholar
  12. Gunsalus RP, Wolfe RS (1978) ATP activation and properties of the methyl coenzyme M reductase system in Methanobacterium thermoautotrophicum. J Bacteriol 135: 851–857PubMedGoogle Scholar
  13. Hartzell PL, Zvilius G, Escalante-Semerena JC, Donnelly MI (1985) Coenzyme F420 dependence of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum. Biochem Biophys Res Commun 133: 884–890PubMedGoogle Scholar
  14. Jacobson FS, Daniels L, Fox JA, Walsh CT, Orme-Johnson WH (1982) Purification and properties of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum. J Biol Chem 257: 3385–3388PubMedGoogle Scholar
  15. Jin SLC, Blanchard DK, Chen JS (1983) Two hydrogenases with distinct electron-carrier specificity and subunit composition in Methanobacterium formicicum Biochim Biophys Acta 748: 8–20Google Scholar
  16. Jones RW, Gray TA, Garland PB (1976) A study of the permeability of the cytoplasmic membrane of Escherichia coli to reduced and oxidized benzyl viologen and methyl viologen cations: complications in the use of viologens as redox mediators for membrane-bound enzymes. Biochem Soc Trans 4: 671–673PubMedGoogle Scholar
  17. Kyte J, Doolittle RF (1982) A simple method for displaying the hydrophatic character of a protein. J Mol Biol 157: 105–132PubMedGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedGoogle Scholar
  19. May HD, Schauer NL, Ferry JG (1986) Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase. J Bacteriol 166: 500–504PubMedGoogle Scholar
  20. May HD, Patel PS, Ferry JG (1988) Effect of molybdenum and tungsten on synthesis and composition of formate dehydrogenase in Methanobacterium formicicum. J Bacteriol 170: 3384–3389PubMedGoogle Scholar
  21. Mayer F, Jussofie A, Salzmann M, Lubben M, Rohde M, Gottschalk G (1987) Immunoelectron microscopic demonstration of ATPase on the cytoplasmic membrane of the methanogenic bacterium strain Gö1. J Bacteriol 169: 2307–2309PubMedGoogle Scholar
  22. Muth E (1988) Localization of the F420-reducing hydrogenase in Methanococcus voltae by immuno-gold technique. Arch Microbiol 150: 205–207Google Scholar
  23. Muth E, Mörschel E, Klein A (1987) Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae. Eur J Biochem 169: 571–577PubMedGoogle Scholar
  24. Nagle Jr DP, Wolfe RS (1983) Component A of the methyl coenzyme M methylreductase system of Methanobacterium: resolution into four components. Proc Natl Acad Sci USA 80: 2151–2155PubMedGoogle Scholar
  25. Nelson MJK, Brown DP, Ferry JG (1984) FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicum. Biochem Biophys Res Commun 120: 775–781PubMedGoogle Scholar
  26. Ossmer R, Mund T, Hartzell P, Konheiser U, Kohring GW, Klein A, Wolfe RS, Gottschalk G, Mayer F (1986) Immunocytochemical localization of component C of the methylreductase system in Methanococcus voltae and Methanobacterium thermoautotrophicum. Proc Natl Acad Sci USA 83: 5789–5792Google Scholar
  27. Patel PS, Ferry JG (1988) Characterization of the upstream region of the formate dehydrogenase operon of Methanobacterium formicicum. J Bacteriol 170: 3390–3395PubMedGoogle Scholar
  28. Schauer NL, Ferry JG (1980) Metabolism of formate in Methanobacterium formicicum. J Bacteriol 142: 800–807PubMedGoogle Scholar
  29. Schauer NL, Ferry JG (1982) Properties of formate dehydrogenase in Methanobacterium formicicum. J Bacteriol 150: 1–7 (Erratum 151: 1642)PubMedGoogle Scholar
  30. Schauer NL, Ferry JG (1983) FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum. J Bacteriol 155: 467–472PubMedGoogle Scholar
  31. Schauer NL, Ferry JG (1986) Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum. J Bacteriol 165: 405–411PubMedGoogle Scholar
  32. Schauer NL, Ferry JG, Honek JF, Orme-Johnson WH, Walsh CT (1986) Mechanistic studies of the coenzyme F420-reducing formate dehydrogenase from Methanobacterium formicicum. Biochemistry 25: 7163–7168PubMedGoogle Scholar
  33. Shuber AP, Orr EC, Recny MA, Schendel PF, May HD, Schauer NL, Ferry JG (1986) Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum. J Biol Chem 261: 12942–12947PubMedGoogle Scholar
  34. Sprott GD, Shaw KM, Beveridge TJ (1987) Properties of the particulate enzyme F420-reducing hydrogenase isolated from Methanospirillum hungatei. Can J Microbiol 33: 896–904Google Scholar
  35. Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354PubMedGoogle Scholar
  36. Yamazaki S (1982) A selenium-containing hydrogenase from Methanococcus vannielii. J Biol Chem 257: 7926–7929PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • S. F. Baron
    • 1
  • D. S. Williams
    • 2
  • H. D. May
    • 1
  • P. S. Patel
    • 1
  • H. C. Aldrich
    • 2
  • J. G. Ferry
    • 1
  1. 1.Department of Anaerobic MicrobiologyVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  2. 2.Department of Microbiology and Cell SciencesUniversity of FloridaGainesvilleUSA

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