Heiden S, Hedderich R, Setzke E, Thauer R K
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität Marburg, Germany.
Eur J Biochem. 1993 Apr 1;213(1):529-35. doi: 10.1111/j.1432-1033.1993.tb17791.x.
The reduction of CoM-S-S-HTP, the heterodisulfide of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP), with H2 is an energy-conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane-bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non-heme iron and acid-labile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme-derived peroxidase activity indicating that this polypeptide is the cytochrome b. The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM-S-S-HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 mumol.min-1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM-S-S-HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM-S-S-HTP and re-reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions. The findings suggest that the H2:heterodisulfide oxidoreductase complex is composed of a F420-non-reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM-S-S-HTP reduction with H2.
辅酶M（H-S-CoM）和N-7-巯基庚酰苏氨酸磷酸（H-S-HTP）的异二硫化合物CoM-S-S-HTP与H2的还原反应是产甲烷古菌中一个节约能量的步骤。我们在此报告，在巴氏甲烷八叠球菌中，该反应由一种膜结合多酶复合物催化，该复合物被命名为H2：异二硫化合物氧化还原酶复合物，已被纯化至表观均一。发现该制剂由9种表观分子量分别为46 kDa、39 kDa、28 kDa、25 kDa、23 kDa、21 kDa、20 kDa、16 kDa和15 kDa的多肽组成，每毫克蛋白质含有3.2 nmol细胞色素b、70至80 nmol非血红素铁和酸不稳定硫、5 nmol镍以及0.6 nmol黄素腺嘌呤二核苷酸。23 kDa的多肽具有血红素衍生的过氧化物酶活性，表明该多肽是细胞色素b。纯化的H2：异二硫化合物氧化还原酶复合物催化CoM-S-S-HTP与H2的还原反应，比活性为6 U/mg蛋白质（1 U = 1 μmol·min-1），催化苄基紫精与H2的还原反应，比活性为66 U/mg蛋白质，催化CoM-S-S-HTP与苄基紫精的还原反应，比活性为66 U/mg蛋白质，以及催化CoM-S-S-HTP与还原型苄基紫精的HTP还原反应，比活性为24 U/mg蛋白质。该复合物不介导辅酶F420与H2的还原反应，也不介导还原型辅酶F420与CoM-S-S-HTP的氧化反应。酶复合物中还原型细胞色素b可被CoM-S-S-HTP氧化，并被H2重新还原。在我们的实验条件下，细胞色素氧化和还原的比速率太高而无法分辨。这些发现表明，H2：异二硫化合物氧化还原酶复合物由一种不还原F420的氢化酶、一种细胞色素b和异二硫化合物还原酶组成，并且细胞色素b是参与CoM-S-S-HTP与H2还原反应的电子传递链中的一种氧化还原载体。
