Interaction of purified NDH-1 from Escherichia coli with ubiquinone analogues.
The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.
David
Pamela
P
Helsinki Bioenergetics Group, Biotechnology Institute, Biocenter 2, University of Helsinki, Helsinki, Finland.
Baumann
Marc
M
Wikström
Mårten
M
Finel
Moshe
M
D001426:Bacterial Proteins / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange / Q000379:methods*; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009245:NADH Dehydrogenase / Q000172:deficiency; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009994:Osmolar Concentration; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry
D001426:Bacterial Proteins; D014451:Ubiquinone; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D009247:NADH, NADPH Oxidoreductases; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; C025204:Ubiquinone Q2; C025203:Ubiquinone Q1