Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles

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Abstract
Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified E. coli strains, were characterized. Enzyme preparations contained 70% (wt/wt) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and .apprx. 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme contained only low levels of Fe and acid-labile S, indicating the absence of Fe-S clusters. No Cu, Mo, W, or covalently bound P was detected and no evidence for other chromophores was obtained from visible and UV 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 MW 47,000 and the N- and C-terminal cyanogen bromide peptide were identified. The pure enzyme reconstituted membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.