Kurki S, Zickermann V, Kervinen M, Hassinen I, Finel M
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, Helsinki, Finland.
Biochemistry. 2000 Nov 7;39(44):13496-502. doi: 10.1021/bi001134s.
Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.
对来自牛线粒体和土壤细菌反硝化副球菌的膜样品中的H(+)-转运NADH：泛醌还原酶（复合体I）的稳态动力学进行了分析。在这两种酶中，在膜脂相中，四种不同泛醌类似物的计算K(m)值都在毫摩尔范围内。受体疏水侧链的结构和大小都影响其对复合体I的亲和力。牛复合体I的ND1亚基是一种线粒体编码的蛋白质，它能与抑制剂二环己基碳二亚胺（DCCD）共价结合[矢木和哈泰菲（1988年）《生物化学杂志》263, 16150 - 16155]。反硝化副球菌复合体I的NQO8亚基是ND1的同源物，其中三个可能结合DCCD的保守Glu残基，即E158、E212和E247，被替换为Asp或Gln，对于E212还替换为Val。然而，所得突变体的DCCD敏感性不受这些突变的影响。另一方面，突变体的泛醌还原酶活性发生了改变，并且这些突变改变了复合体I与短链泛醌的相互作用。讨论了这些结果对该酶中泛醌还原位点位置的影响。
