Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.
The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.
Yu
L
L
Department of Biochemistry, Oklahoma State University, Stillwater 74078.
Yu
C A
CA
D000906:Antibodies; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D002846:Chromatography, Affinity; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D014450:Electron Transport Complex III