Abstract
The distribution of the F420-reactive and F420-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F420-nonreactive enzyme. The membrane-bound F420-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 μmol H2 oxidized · min-1 · mg protein-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F420-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F420-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.
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Abbreviations
- \(\Delta \tilde \mu H + \) :
-
transmembrane electrochemical gradient of H-
- CoM-SH:
-
2-mercaptoethanesulfonate
- F420 :
-
(N-l-lactyl-γ-l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphate
- F420H2 :
-
reduced F420
- HTP-SH:
-
7-mercaptoheptanoylthreonine phosphate
- Mb. :
-
Methanobacterium
- PMSF:
-
phenylmethyl-sulfonylfluoride
- Cl3AcOH:
-
trichloroacetic acid
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Deppenmeier, U., Blaut, M., Schmidt, B. et al. Purification and properties of a F420-nonreactive, membrane-bound hydrogenase from Methanosarcina strain Gö1. Arch. Microbiol. 157, 505–511 (1992). https://doi.org/10.1007/BF00276770
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DOI: https://doi.org/10.1007/BF00276770