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 ≥ 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.