1887

Abstract

is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO by reducing H to H. A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from . A ferredoxin isolated from was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron–sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H)] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H). These results indicate a regulation in response to the p(H).

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27159-0
2004-07-01
2019-09-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502451.html?itemId=/content/journal/micro/10.1099/mic.0.27159-0&mimeType=html&fmt=ahah

References

  1. Adams, M. W. ( 1990; ). The structure and mechanism of iron-hydrogenases. Biochim Biophys Acta 1020, 115–145.[CrossRef]
    [Google Scholar]
  2. Albracht, S. P. & Hedderich, R. ( 2000; ). Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH-ubiquinone oxidoreductase (Complex I). FEBS Lett 485, 1–6.[CrossRef]
    [Google Scholar]
  3. Amend, J. P. & Plyasunov, A. V. ( 2001; ). Carbohydrates in thermophile metabolism: calculation of the standard molal thermodynamic properties of aqueous pentoses and hexoses at elevated temperatures and pressures. Geochim Cosmochim Acta 65, 3901–3917.[CrossRef]
    [Google Scholar]
  4. Amend, J. P. & Shock, E. L. ( 2001; ). Energetics of overall metabolic reactions of thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol Rev 25, 175–243.[CrossRef]
    [Google Scholar]
  5. Bao, Q., Tian, Y., Li, W. & 18 other authors ( 2002; ). A complete sequence of the T. tengcongensis genome. Genome Res 12, 689–700.[CrossRef]
    [Google Scholar]
  6. Beinert, H. & Albracht, S. P. J. ( 1982; ). New insights, ideas and unanswered questions concerning iron-sulfur clusters in mitochondria. Biochim Biophys Acta 683, 245–277.[CrossRef]
    [Google Scholar]
  7. Blokesch, M., Paschos, A., Theodoratou, E., Bauer, A., Hube, M., Huth, S. & Bock, A. ( 2002; ). Metal insertion into NiFe-hydrogenases. Biochem Soc Trans 30, 674–680.
    [Google Scholar]
  8. Böhm, R., Sauter, M. & Böck, A. ( 1990; ). Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase components. Mol Microbiol 4, 231–243.[CrossRef]
    [Google Scholar]
  9. Bott, M. & Thauer, R. K. ( 1989; ). Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri. Eur J Biochem 179, 469–472.[CrossRef]
    [Google Scholar]
  10. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  11. Burdette, D. S. & Zeikus, J. G. ( 1994; ). Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39e and characterisation of the secondary-alcohol dehydrogenase as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioesterase. Biochem J 302, 163–170.
    [Google Scholar]
  12. Burdette, D. S., Vieille, C. & Zeikus, J. G. ( 1996; ). Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme. Biochem J 316, 115–122.
    [Google Scholar]
  13. Burdette, D. S., Jung, S. H., Shen, G. J., Hollingsworth, R. I. & Zeikus, J. G. ( 2002; ). Physiological function of alcohol dehydrogenases and long-chain (C-30) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus. Appl Environ Microbiol 68, 1914–1918.[CrossRef]
    [Google Scholar]
  14. Chen, J.-S. & Blanchard, D. K. ( 1979; ). A simple hydrogenase-linked assay for ferredoxin and flavodoxin. Anal Biochem 93, 216–222.[CrossRef]
    [Google Scholar]
  15. De Luca, G., Asso, M., Belaich, J. P. & Dermoun, Z. ( 1998; ). Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. Biochemistry 37, 2660–2665.[CrossRef]
    [Google Scholar]
  16. Dorn, M., Andreesen, J. R. & Gottschalk, G. ( 1978; ). Fermentation of fumarate and l-malate by Clostridium formicoaceticum. J Bacteriol 133, 26–32.
    [Google Scholar]
  17. Fox, J. D., He, Y., Shelver, D., Roberts, G. P. & Ludden, P. W. ( 1996b; ). Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. J Bacteriol 178, 6200–6208.
    [Google Scholar]
  18. Friedrich, T. & Scheide, D. ( 2000; ). The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases. FEBS Lett 479, 1–5.[CrossRef]
    [Google Scholar]
  19. Friedrich, T. & Weiss, H. ( 1997; ). Modular evolution of the respiratory NADH:ubiquinone oxidoreductase and the origin of its modules. J Theor Biol 187, 529–540.[CrossRef]
    [Google Scholar]
  20. Gottschalk, G. ( 1986; ). Bacterial Metabolism. New York: Springer.
  21. Hedderich, R. ( 2004; ). Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I. J Bioenerg Biomembr 36, 65–75.[CrossRef]
    [Google Scholar]
  22. Jungermann, K., Thauer, R. K., Leimenstoll, G. & Decker, K. ( 1973; ). Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia. Biochim Biophys Acta 305, 268–280.[CrossRef]
    [Google Scholar]
  23. Kerby, R. L., Ludden, P. W. & Roberts, G. P. ( 1995; ). Carbon monoxide-dependent growth of Rhodospirillum rubrum. J Bacteriol 177, 2241–2244.
    [Google Scholar]
  24. Künkel, A., Vorholt, J. A., Thauer, R. K. & Hedderich, R. ( 1998; ). An Escherichia coli hydrogenase-3-type hydrogenase in methanogenic archaea. Eur J Biochem 252, 467–476.[CrossRef]
    [Google Scholar]
  25. Kunst, A., Draeger, B. & Ziegenhorn, J. ( 1981; ). Colorimetric methods with glucose oxidase and peroxidase. In Methods of Enzymatic Analysis, pp. 178–185. Edited by H. U. Bergmeyer. Weinheim: Verlag Chemie.
  26. Malki, S., Saimmaime, I., De Luca, G., Rousset, M., Dermoun, Z. & Belaich, J. P. ( 1995; ). Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans. J Bacteriol 177, 2628–2636.
    [Google Scholar]
  27. Meuer, J., Bartoschek, S., Koch, J., Künkel, A. & Hedderich, R. ( 1999; ). Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri. Eur J Biochem 265, 325–335.[CrossRef]
    [Google Scholar]
  28. Meuer, J., Kuettner, H. C., Zhang, J. K., Hedderich, R. & Metcalf, W. W. ( 2002; ). Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation. Proc Natl Acad Sci U S A 99, 5632–5637.[CrossRef]
    [Google Scholar]
  29. Pan, G., Menon, A. L. & Adams, M. W. ( 2003; ). Characterization of a [2Fe-2S] protein encoded in the iron-hydrogenase operon of Thermotoga maritima. J Biol Inorg Chem 8, 469–474.
    [Google Scholar]
  30. Peters, J. W., Lanzilotta, W. N., Lemon, B. J. & Seefeldt, L. C. ( 1998; ). X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1·8 angstrom resolution. Science 282, 1853–1858.[CrossRef]
    [Google Scholar]
  31. Sapra, R., Verhagen, M. & Adams, M. W. W. ( 2000; ). Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 182, 3423–3428.[CrossRef]
    [Google Scholar]
  32. Sapra, R., Bagramyan, K. & Adams, M. W. ( 2003; ). A simple energy-conserving system: proton reduction coupled to proton translocation. Proc Natl Acad Sci U S A 100, 7545–7550.[CrossRef]
    [Google Scholar]
  33. Sauter, M., Böhm, R. & Böck, A. ( 1992; ). Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Mol Microbiol 6, 1523–1532.[CrossRef]
    [Google Scholar]
  34. Sawers, G. ( 1994; ). The hydrogenases and formate dehydrogenases of Escherichia coli. Antonie Van Leeuwenhoek 66, 57–88.[CrossRef]
    [Google Scholar]
  35. Schmitz, R. A., Daniel, R., Deppenmeier, U. & Gottschalk, G. ( 2003; ). The anaerobic way of life. In The Prokaryotes: an Evolving Electronic Resource for the Microbiological Community (http://141.150.157.117 : 8080/prokPUB/index.htm). Edited by M. Dworkin and others. New York: Springer.
  36. Schröder, C., Selig, M. & Schönheit, P. ( 1994; ). Glucose fermentation to acetate, CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Emden-Meyerhof pathway. Arch Microbiol 161, 460–470.
    [Google Scholar]
  37. Schwarz, E. & Friedrich, B. ( 2003; ). The H2-metabolizing prokaryotes. In The Prokaryotes: an Evolving Electronic Resource for the Microbiological Community (http://141.150.157.117 : 8080/prokPUB/index.htm). Edited by M. Dworkin and others. New York: Springer.
  38. Silva, P. J., van den Ban, E. C., Wassink, H., Haaker, H., de Castro, B., Robb, F. T. & Hagen, W. R. ( 2000; ). Enzymes of hydrogen metabolism in Pyrococcus furiosus. Eur J Biochem 267, 6541–6551.[CrossRef]
    [Google Scholar]
  39. Soboh, B., Linder, D. & Hedderich, R. ( 2002; ). Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans. Eur J Biochem 269, 5712–5721.[CrossRef]
    [Google Scholar]
  40. Svetlichny, V. A., Sokolova, T. G., Gerhardt, M., Ringpfeil, M., Kostrikina, N. A. & Zavarzin, G. A. ( 1991; ). Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing thermophilic anaerobic bacterium from hypothermal environments of Kunashir island. Syst Appl Microbiol 14, 254–260.[CrossRef]
    [Google Scholar]
  41. Tersteegen, A. & Hedderich, R. ( 1999; ). Methanobacterium thermoautotrophicum encodes two multi-subunit membrane-bound [NiFe] hydrogenases. Transcription of the operons and sequence analysis of the deduced proteins. Eur J Biochem 264, 930–943.[CrossRef]
    [Google Scholar]
  42. Tewes, F. J. & Thauer, R. K. ( 1980; ). Regulation of ATP-synthesis in glucose fermenting bacteria involved in interspecies hydrogen transfer. In Anaerobes and Anaerobic Infections, pp. 97–104. Edited by G. Gottschalk. Stuttgart & New York: Gustav Fischer Verlag.
  43. Thauer, R. K., Jungermann, K. & Decker, K. ( 1977; ). Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41, 100–180.
    [Google Scholar]
  44. van Niel, E. W., Claassen, P. A. & Stams, A. J. ( 2003; ). Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 81, 255–262.[CrossRef]
    [Google Scholar]
  45. Verhagen, M. F., O'Rourke, T. & Adams, M. W. ( 1999; ). The hyperthermophilic bacterium, Thermotoga maritima, contains an unusually complex iron-hydrogenase: amino acid sequence analyses versus biochemical characterization. Biochim Biophys Acta 1412, 212–229.[CrossRef]
    [Google Scholar]
  46. Verhagen, M. F., O'Rourke, T. W., Menon, A. L. & Adams, M. W. ( 2001; ). Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima. Biochim Biophys Acta 1505, 209–219.[CrossRef]
    [Google Scholar]
  47. Vignais, P. M., Billoud, B. & Meyer, J. ( 2001; ). Classification and phylogeny of hydrogenases. FEMS Microbiol Rev 25, 455–501.[CrossRef]
    [Google Scholar]
  48. Wang, J., Xue, Y., Feng, X. & 11 other authors ( 2004; ). An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions. Proteomics 4, 136–150.[CrossRef]
    [Google Scholar]
  49. Weidner, U., Geier, S., Ptock, A., Friedrich, T., Leif, H. & Weiss, H. ( 1993; ). The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol 233, 109–122.[CrossRef]
    [Google Scholar]
  50. West, A. H. & Stock, A. M. ( 2001; ). Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci 26, 369–376.[CrossRef]
    [Google Scholar]
  51. Xue, Y., Xu, Y., Liu, Y., Ma, Y. & Zhou, P. ( 2001; ). Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 51, 1335–1341.
    [Google Scholar]
  52. Yano, T. & Ohnishi, T. ( 2001; ). The origin of cluster N2 of the energy-transducing NADH-quinone oxidoreductase: comparisons of phylogenetically related enzymes. J Bioenerg Biomembr 33, 213–222.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27159-0
Loading
/content/journal/micro/10.1099/mic.0.27159-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error