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Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH‐2) and analysis of its interaction with ubiquinone analogues
Author(s) -
Björklöf Katja,
Zickermann Volker,
Finel Moshe
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01130-3
Subject(s) - nadh dehydrogenase , escherichia coli , biochemistry , enzyme , chemistry , dehydrogenase , reductase , membrane , transmembrane protein , dihydrolipoamide dehydrogenase , electron transport chain , membrane protein , receptor , protein subunit , gene
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.