title: The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization.
abstract: The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.
authors.affiliation: Institut für Mikrobiologie der Universität Göttingen, Federal Republic of Germany.
authors.forename: K
authors.initials: K
authors.lastname: Knüttel
authors.forename: K
authors.initials: K
authors.lastname: Schneider
authors.forename: H G
authors.initials: HG
authors.lastname: Schlegel
authors.forename: A
authors.initials: A
authors.lastname: Müller
mesh_terms: D000421:Alcaligenes / Q000201:enzymology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D006864:Hydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D007526:Isoelectric Point; D008670:Metals / Q000032:analysis; D008970:Molecular Weight; D010231:Paracoccus denitrificans / Q000201:enzymology*; D012995:Solubility
chemical_list: D008670:Metals; D006864:Hydrogenase