Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles.
Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.
D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003579:Cytochrome Reductases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005415:Flavins / Q000032:analysis; D007501:Iron / Q000032:analysis; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D009245:NADH Dehydrogenase / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010101:Oxygen Consumption; D010957:Plasmids; D011485:Protein Binding; D013045:Species Specificity; D013440:Sulfides / Q000032:analysis; D014451:Ubiquinone / Q000032:analysis
D000596:Amino Acids; D005415:Flavins; D009097:Multienzyme Complexes; D013440:Sulfides; D014451:Ubiquinone; D007501:Iron; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase