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

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-l 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 accepters. High V-max and low KM 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, (C) 2000 Federation of European Biochemical Societies.