Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli.
Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component. The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E. coli bd complex.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J F
JF
Kolonay
F
F
Moshiri
R B
RB
Gennis
T M
TM
Kaysser
R J
RJ
Maier
D000332:Aerobiosis; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010101:Oxygen Consumption; D013053:Spectrophotometry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D014451:Ubiquinone; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; C003741:ubiquinol