Schulz H, Albracht S P, Coremans J M, Fuchs G
Abteilung Angewandte Mikrobiologie, Universität Ulm, Federal Republic of Germany.
Eur J Biochem. 1988 Feb 1;171(3):589-97. doi: 10.1111/j.1432-1033.1988.tb13829.x.
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.
嗜热自养甲烷杆菌的细胞质膜不含细胞色素，但含有一种分子量约为33 kDa的类咕啉蛋白，在用10 mg Triton X - 100/mg蛋白处理后，该蛋白存在于一个约500 kDa的蛋白复合物中。从1 g干细胞中洗涤得到的膜含有约70 nmol的钴胺素因子III（5 - 羟基苯并咪唑基钴胺素），这是唯一的类咕啉。含类咕啉的蛋白复合物被纯化并研究了其一些性质。根据几个标准，它是一种整合膜蛋白复合物。类咕啉 - 蛋白复合物在经过约100倍纯化后，在非变性聚丙烯酰胺凝胶电泳（native PAGE）上呈现单一条带，分子量仍约为500 kDa。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中观察到几个亚基：除了约33 kDa的携带类咕啉的亚基外，还存在约28 kDa、26 kDa以及可能23 kDa的其他多肽。1摩尔纯化的500 kDa蛋白复合物含有大于或等于8摩尔的钴胺素因子III。据估计，类咕啉 - 蛋白复合物占嗜热自养甲烷杆菌膜蛋白的8%。氧化态蛋白的可见光谱在547 nm、511 nm处有吸收最大值，在468 nm处有一个肩峰，在用连二亚硫酸盐还原后消失。该转变的中点电位约为 - 145 mV（pH 7）。通过电子顺磁共振（EPR）观察到在 - 50 mV至 - 350 mV范围内有Co2 +信号，最大值在 - 200 mV左右。讨论了在低氧化还原电位下Co2 +信号消失的可能原因。Co2 +信号的线形与游离类咕啉中Co2 +的线形相似。5 mM二硫苏糖醇还原也可引发Co2 +信号。从类咕啉膜蛋白的氧化还原性质可以预期，在体内钴可能会被还原和再氧化。讨论了其在产甲烷细菌代谢中作为电子介导膜蛋白的可能功能。
