Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH‐2) and analysis of its interaction with ubiquinone analogues

FEBS Letters

Finel, Moshe¹  Björklöf, Katja¹  Zickermann, Volker¹ 

[1] Helsinki Bioenergetics Group, Department of Medical Chemistry, University of Helsinki, P.O. Box 8, Siltavuorenpenger 10, FIN-00014 Helsinki, Finland

关键词: Deamino-NADH;    Idebenone;    NDH-2;    NADH:quinone reductase;    Ubiquinone;    NAD(P)H-(disulfide)-oxidoredeductase;    DB;    decylbenzoquinone (decylubiquinone);    DCIP;    dichlorophenolindophenol;    Idebenone;    hydroxidecyl benzoquinone;    IPTG;    isopropylthio-β-D-galactoside;    NDH-1;    H+-translocating NADH:quinone oxidoreductase (Complex I);    NDH-2;    the ‘alternative’;    non-H+-pumping NADH:quinone reductase;    PMSF;    phenylmethylsulfonyl fluoride;    Q1;    ubiquinone-1;    Q2;    ubiquinone-2;   

DOI  :  10.1016/S0014-5793(00)01130-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The NADH:ubiquinone reductase (NDH-2) of Escherichia coli was expressed as a His-tagged protein, extracted from the membrane fraction using detergent and purified by chromatography. The His-tagged NDH-2 was highly active and catalyzed NADH oxidation by ubiquinone-1 at rates over two orders of magnitude higher than previously reported. The purified, His-tagged NDH-2, like native NDH-2, did not oxidize deamino-NADH. Steady-state kinetics were used to analyze the enzyme's activity in the presence of different electron acceptors. High V _max and low K _m values were only found for hydrophobic ubiquinone analogues, particularly ubiquinone-2. These findings strongly support the notion that NDH-2 is a membrane bound enzyme, despite the absence of predicted transmembrane segments in its primary structure. The latter observation is in agreement with possible evolutionary relation between NDH-2 and water-soluble enzymes such as dihydrolipoamide dehydrogenase. There is currently no clear indication of how NDH-2 binds to biological membranes.

【 授权许可】

Unknown