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. 1979 Jul;139(1):231–238. doi: 10.1128/jb.139.1.231-238.1979

Solubilization and Properties of a Particulate Hydrogenase from Methanobacterium Strain G2R†

R C McKellar 1,‡, G D Sprott 1
PMCID: PMC216850  PMID: 37236

Abstract

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-β-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77°C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5′-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

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Selected References

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  1. Ackrell B. A., Asato R. N., Mower H. F. Multiple forms of bacterial hydrogenases. J Bacteriol. 1966 Oct;92(4):828–838. doi: 10.1128/jb.92.4.828-838.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams M. W., Hall D. O. Isolation of the membrane-bound hydrogenase from Rhodospirillum rubrum. Biochem Biophys Res Commun. 1977 Jul 25;77(2):730–737. doi: 10.1016/s0006-291x(77)80039-9. [DOI] [PubMed] [Google Scholar]
  3. Aspen A. J., Wolin M. J. Solubilization and reconstitution of a particulate hydrogenase from Vibrio succinogenes. J Biol Chem. 1966 Sep 25;241(18):4152–4156. [PubMed] [Google Scholar]
  4. Baron C., Thompson T. E. Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. Biochim Biophys Acta. 1975 Mar 25;382(3):276–285. doi: 10.1016/0005-2736(75)90270-9. [DOI] [PubMed] [Google Scholar]
  5. Bell G. R., LeGall L., Peck H. D. Evidence for the periplasmic location of hydrogenase in Desulfovibrio gigas. J Bacteriol. 1974 Nov;120(2):994–997. doi: 10.1128/jb.120.2.994-997.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bell G. R., Lee J. P., Peck H. D., Jr, Gall J. L. Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors. Biochimie. 1978;60(3):315–320. doi: 10.1016/s0300-9084(78)80828-1. [DOI] [PubMed] [Google Scholar]
  7. Cheeseman P., Toms-Wood A., Wolfe R. S. Isolation and properties of a fluorescent compound, factor 420 , from Methanobacterium strain M.o.H. J Bacteriol. 1972 Oct;112(1):527–531. doi: 10.1128/jb.112.1.527-531.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen J. S., Mortenson L. E. Purification and properties of hydrogenase from Clostridium pasteurianum W5. Biochim Biophys Acta. 1974 Dec 18;371(2):283–298. doi: 10.1016/0005-2795(74)90025-7. [DOI] [PubMed] [Google Scholar]
  9. Daniels L., Fuchs G., Thauer R. K., Zeikus J. G. Carbon monoxide oxidation by methanogenic bacteria. J Bacteriol. 1977 Oct;132(1):118–126. doi: 10.1128/jb.132.1.118-126.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doddema H. J., Hutten T. J., van der Drift C., Vogels G. D. ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum. J Bacteriol. 1978 Oct;136(1):19–23. doi: 10.1128/jb.136.1.19-23.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erbes D. L., Burris R. H. The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianum. Biochim Biophys Acta. 1978 Jul 7;525(1):45–54. doi: 10.1016/0005-2744(78)90198-5. [DOI] [PubMed] [Google Scholar]
  12. Feigenblum E., Krasna A. I. Solubilization and properties of the hydrogenase of Chromatium. Biochim Biophys Acta. 1970 Feb 11;198(2):157–164. doi: 10.1016/0005-2744(70)90047-1. [DOI] [PubMed] [Google Scholar]
  13. Gogotov I. N., Zorin N. A., Serebriakova L. T., Kondratieva E. N. The properties of hydrogenase from Thiocapsa roseopersicina. Biochim Biophys Acta. 1978 Apr 12;523(2):335–343. doi: 10.1016/0005-2744(78)90036-0. [DOI] [PubMed] [Google Scholar]
  14. Gunsalus R. P., Romesser J. A., Wolfe R. S. Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum. Biochemistry. 1978 Jun 13;17(12):2374–2377. doi: 10.1021/bi00605a019. [DOI] [PubMed] [Google Scholar]
  15. Gunsalus R. P., Wolfe R. S. ATP activation and properties of the methyl coenzyme M reductase system in Methanobacterium thermoautotrophicum. J Bacteriol. 1978 Sep;135(3):851–857. doi: 10.1128/jb.135.3.851-857.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gunsalus R. P., Wolfe R. S. Stimulation of CO2 reduction to methane by methylcoenzyme M in extracts Methanobacterium. Biochem Biophys Res Commun. 1977 Jun 6;76(3):790–795. doi: 10.1016/0006-291x(77)91570-4. [DOI] [PubMed] [Google Scholar]
  17. Hatchikian E. C., Bruschi M., Le Gall J. Characterization of the periplasmic hydrogenase from Desulfovibrio gigas. Biochem Biophys Res Commun. 1978 May 30;82(2):451–461. doi: 10.1016/0006-291x(78)90896-3. [DOI] [PubMed] [Google Scholar]
  18. McBride B. C., Wolfe R. S. A new coenzyme of methyl transfer, coenzyme M. Biochemistry. 1971 Jun 8;10(12):2317–2324. doi: 10.1021/bi00788a022. [DOI] [PubMed] [Google Scholar]
  19. O'Brien R. W., Morris J. G. The Ferredoxin-dependent reduction of chloramphenicol by clostridium acetobutylicum. J Gen Microbiol. 1971 Aug;67(3):265–271. doi: 10.1099/00221287-67-3-265. [DOI] [PubMed] [Google Scholar]
  20. Patel G. B., Khan A. W., Roth L. A. Optimum levels of sulphate and iron for the cultivation of pure cultures of methanogens in synthetic media. J Appl Bacteriol. 1978 Dec;45(3):347–356. doi: 10.1111/j.1365-2672.1978.tb04235.x. [DOI] [PubMed] [Google Scholar]
  21. Patel G. B., Roth L. A., van den Berg L., Clark D. S. Characterization of a strain of Methanospirillum hungatti. Can J Microbiol. 1976 Sep;22(9):1404–1410. doi: 10.1139/m76-208. [DOI] [PubMed] [Google Scholar]
  22. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  23. Probst I., Schlegel H. G. Respiratory components and oxidase activities in Alcaligenes eutrophus. Biochim Biophys Acta. 1976 Aug 13;440(2):412–428. doi: 10.1016/0005-2728(76)90075-x. [DOI] [PubMed] [Google Scholar]
  24. Robertson A. M., Wolfe R. S. ATP requirement for methanogenesis in cell extracts of methanobacterium strain M.o.H. Biochim Biophys Acta. 1969 Dec 30;192(3):420–429. doi: 10.1016/0304-4165(69)90391-2. [DOI] [PubMed] [Google Scholar]
  25. Sauer F. D., Bush R. S., Mahadevan S., Erfle J. D. Methane production by cell-free particulate fraction of rumen bacteria. Biochem Biophys Res Commun. 1977 Nov 7;79(1):124–132. doi: 10.1016/0006-291x(77)90069-9. [DOI] [PubMed] [Google Scholar]
  26. Schink B., Schlegel H. G. Mutants of Alcaligenes eutrophus defective in autotrophic metabolism. Arch Microbiol. 1978 May 30;117(2):123–129. doi: 10.1007/BF00402299. [DOI] [PubMed] [Google Scholar]
  27. Schneider K., Schlegel H. G. Localization and stability of hydrogenases from aerobic hydrogen bacteria. Arch Microbiol. 1977 Apr 1;112(3):229–238. doi: 10.1007/BF00413086. [DOI] [PubMed] [Google Scholar]
  28. Schneider K., Schlegel H. G. Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. Biochim Biophys Acta. 1976 Nov 8;452(1):66–80. doi: 10.1016/0005-2744(76)90058-9. [DOI] [PubMed] [Google Scholar]
  29. Sim E., Vignais P. M. Hydrogenase activity in Paracoccus denitrificans. Partial purification and interaction with the electron transport chain. Biochimie. 1978;60(3):307–314. doi: 10.1016/s0300-9084(78)80827-x. [DOI] [PubMed] [Google Scholar]
  30. Sprott G. D., McKellar R. C., Shaw K. M., Giroux J., Martin W. G. Properties of malate dehydrogenase isolated from Methanospirillum hungatii. Can J Microbiol. 1979 Feb;25(2):192–200. doi: 10.1139/m79-030. [DOI] [PubMed] [Google Scholar]
  31. Thauer R. K., Jungermann K., Decker K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 1977 Mar;41(1):100–180. doi: 10.1128/br.41.1.100-180.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tzeng S. F., Wolfe R. S., Bryant M. P. Factor 420-dependent pyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium. J Bacteriol. 1975 Jan;121(1):184–191. doi: 10.1128/jb.121.1.184-191.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  34. Wolfe R. S. Microbial formation of methane. Adv Microb Physiol. 1971;6:107–146. doi: 10.1016/s0065-2911(08)60068-5. [DOI] [PubMed] [Google Scholar]
  35. Zeikus J. G., Fuchs G., Kenealy W., Thauer R. K. Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum. J Bacteriol. 1977 Nov;132(2):604–613. doi: 10.1128/jb.132.2.604-613.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zeikus J. G. The biology of methanogenic bacteria. Bacteriol Rev. 1977 Jun;41(2):514–541. doi: 10.1128/br.41.2.514-541.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van der Westen H. M., Mayhew S. G., Veeger C. Separation of hydrogenase from intact cells of Desulfovibrio vulgaris. Purification and properties. FEBS Lett. 1978 Feb 1;86(1):122–126. doi: 10.1016/0014-5793(78)80112-4. [DOI] [PubMed] [Google Scholar]

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