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-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.
Björklöf
K
K
Helsinki Bioenergetics Group, Department of Medical Chemistry, University of Helsinki, P.O. Box 8, Siltavuorenpenger 10, FIN-00014, Helsinki, Finland.
Zickermann
V
V
Finel
M
M
D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000378:metabolism; D002846:Chromatography, Affinity; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility; D013379:Substrate Specificity; D013816:Thermodynamics; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism*
D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; D009243:NAD; D014451:Ubiquinone; D009245:NADH Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase