One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556.
Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms. The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme. The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification. In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity. This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon. The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule. The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate.
Kita
K
K
Department of Biochemistry, University of Illinois, Urbana 61801.
Vibat
C R
CR
Meinhardt
S
S
Guest
J R
JR
Gennis
R B
RB
D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange / Q000379:methods; D003573:Cytochrome b Group / Q000302:isolation & purification* / Q000378:metabolism; D042963:Electron Transport Complex II; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry; D013385:Succinate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism
D003573:Cytochrome b Group; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; C020730:cytochrome b566; D010088:Oxidoreductases; C022691:succinate oxidase; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase