Heiden S, Hedderich R, Setzke E, Thauer R K
Max-Planck-Institut für Terrestrische Mikrobiologie, Philipps-Universität, Marburg, Germany.
Eur J Biochem. 1994 Apr 15;221(2):855-61. doi: 10.1111/j.1432-1033.1994.tb18800.x.
Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl beta-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.
异二硫键还原酶催化产甲烷古菌能量守恒电子传递链中的末端步骤。甲醇培养的巴氏甲烷八叠球菌膜部分的异二硫键还原酶活性可被Chaps溶解。在Q-琼脂糖凝胶和Superdex-200上进行色谱分离得到一个高分子质量级分（>700 kDa），该级分被十二烷基-β-D-麦芽糖苷解离。在Q-琼脂糖凝胶上进行色谱分离后，得到一种活性异二硫键还原酶制剂，它仅由表观分子质量分别为46 kDa和23 kDa的两种不同亚基组成。每69 kDa的酶含有0.6 mol细胞色素b、0.2 mol FAD、20 mol非血红素铁和20 mol酸不稳定硫。23 kDa的亚基具有血红素衍生的过氧化物酶活性，表明该多肽就是细胞色素b。纯化后的酶含有还原形式的细胞色素b。加入辅酶M和N-7-巯基庚酰苏氨酸磷酸的异二硫键后，细胞色素立即被氧化，这表明细胞色素b作为异二硫键还原的电子供体。
