The respiratory chain NADH dehydrogenase of Escherichia coli. Isolation of an NADH:quinone oxidoreductase from membranes and comparison with the membrane-bound NADH:dichlorophenolindophenol oxidoreductase.

An NADH:quinone oxidoreductase that accounts for all of the NADH oxidase activity of Escherichia coli has been extracted and purified from membrane preparations.Quinone reduction in intact membranes, or after extraction with Triton X-100, is dependent upon NADH, and NADPH is ineffective.The enzyme was purified 100-fold from membrane extracts by chromatography on AMP-Sepharose, DEAE-Sephacel, and Bio-Gel A-1.5M in Triton X-100-containing buffers.This reflects a 2,000-fold purification from whole cells, with a 5% yield.The enzyme has an apparent K,,, for: NADH of 50 pM, decylbenzoquinone of 12 p ~, ubiquinone-3 of 40 pM, and dichlorophenolindophenol of 10 p~ (the latter substrate exhibits sigmoidal kinetics).The highest V,, occurs with decylbenzoquinone (75 units/mg) and ubiquinone-3 (50 units/mg); the NADH:quinone oxidoreductase is less active with dichlorophenolindophenol (V,, = 7.9 units/mg) and has relatively low activity with ferricyanide, menadione, and menaquinone-3.The enzyme responds in a sigmoidal fashion to NADH, with half-maximal activity at 50 PM and substrate inhibition over 200 p .Triton X-100 acts synergistically with cardiolipin to stimulate the enzyme, with optimal activity found at 0.0125% TX-100, where a ?-fold stimulation by cardiolipin occurs.Deoxycholate inhibits the enzyme both in the presence and absence of lipid.Both the NADH oxidase of intact membranes and the purified NADH: quinone oxidoreductase are inhibited at similar concentrations of 3-n-dodecylmercapto-2-hydroxy-1,4-naphthoquinone.In some preparations up to 95% of the protein of the quinone reductase may be accounted for by a polypeptide of 46,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.By atomic absorption spectroscopy there is about 1 mol of Fe/46,000 daltons of protein; since the enzyme has no absorption bands in the region 350-700 nm, it may be a non-heme Fe protein.The NADH:quinone oxidoreductase differs from a purified E. coli membrane-bound NADH:dichlorophenolindophenol (DCIP) oxidoreductase (Dancey, G. F.,