title: Purification and some properties of the corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum.
abstract: The cytoplasmic membrane of the methanogenic archaebacterium Methanobacterium thermoautotrophicum does not contain cytochromes, but did contain a corrinoid protein of molecular mass about 33 kDa which, after treatment with 10 mg Triton X-100/mg protein, was contained in a protein complex of about 500 kDa. Washed membranes from 1 g dry cells contained about 70 nmol of the cobamide factor III (5-hydroxybenzimidazolyl cobamide) as the sole corrinoid. The corrinoid-containing protein complex was purified and some of its properties were studied. According to several criteria it is an integral membrane protein complex. The corrinoid-protein complex, after about 100-fold purification, gave a single band on native PAGE and still had molecular mass of about 500 kDa. In SDS-PAGE several subunits were observed: in addition to the corrinoid-carrying subunit of about 33 kDa, other polypeptides of approximately 28 kDa, 26 kDa, and possibly 23 kDa were present. One mole of the purified 500-kDa protein complex contained greater than or equal to eight moles of the cobamide factor III. It was estimated that the corrinoid-protein complex accounts for 8% of the membrane protein of M. thermoautotrophicum. The visible spectrum of the oxidized protein exhibited absorbance maxima at 547 nm, 511 nm, and a shoulder at 468 nm, which disappeared upon reduction with dithionite. The midpoint potential of this transition was around -145 mV (pH 7). With EPR a Co2+ signal was observed within -50 mV and -350 mV with a maximum around -200 mV. Possible reasons for the disappearance of the Co2+ signal at low redox potentials are discussed. The line shape of the Co2+ signal was similar to that of Co2+ in free corrinoids. The signal of Co2+ could also be evoked by reduction with 5 mM dithiothreitol. From the redox properties of the corrinoid membrane protein it may be expected that in vivo the cobalt may become reduced and reoxidized. Its possible function as an electron-mediating membrane protein in the metabolism of methanogenic bacteria is discussed.
authors.affiliation: Abteilung Angewandte Mikrobiologie, Universität Ulm, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Schulz
authors.forename: S P
authors.initials: SP
authors.lastname: Albracht
authors.forename: J M
authors.initials: JM
authors.lastname: Coremans
authors.forename: G
authors.initials: G
authors.lastname: Fuchs
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification*; D045728:Corrinoids; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D014805:Vitamin B 12 / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D045728:Corrinoids; D008565:Membrane Proteins; D014805:Vitamin B 12