Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain.
The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.
Hayashi
M
M
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Miyoshi
T
T
Takashina
S
S
Unemoto
T
T
D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010101:Oxygen Consumption; D011808:Quinone Reductases / Q000378:metabolism
D009247:NADH, NADPH Oxidoreductases; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; C014227:ferricyanide reductase