Large-scale purification and characterization of a highly active four-subunit cytochrome bc1 complex from Rhodobacter sphaeroides.
A highly active, large-scale preparation of ubiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enzyme was solubilized from chromatophores by using dodecyl maltoside in the presence of glycerol and was purified by anion-exchange and gel filtration chromatography. The procedure yields 35 mg of pure bc1 complex from 4.5 g of membrane protein, and its consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250-350 (mumol of cyt c reduced).(mumol of cyt c1)-1.s-1. The turnover number is at least double that of the best preparation reported in the literature [Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. C., & Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241]. The scale is increased 25-fold, and the yield is markedly improved by using this protocol. Four polypeptide subunits were observed by SDS-PAGE, with Mr values of 40K, 34K, 24K, and 14K. N-Terminal amino acid sequences were obtained for cytochrome c1, the iron-sulfur protein subunit, and for cytochrome b and were identical with the expected protein sequences deduced from the DNA sequence of the fbc operon, with the exceptions that a 22-residue fragment is processed off of the N-terminus of cytochrome c1 and the N-terminal methionine residue is cleaved off both the b cytochrome and iron-sulfur protein subunits. Western blotting experiments indicate that subunit IV is not a contaminating light-harvesting complex polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Andrews
K M
KM
Department of Biochemistry, University of Illinois, Urbana 61801.
Crofts
A R
AR
Gennis
R B
RB
D000595:Amino Acid Sequence; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D016227:Benzoquinones; D014450:Electron Transport Complex III / Q000302:isolation & purification*; D006418:Heme; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011809:Quinones; D012242:Rhodobacter sphaeroides / Q000187:drug effects / Q000201:enzymology*; D013053:Spectrophotometry
D016227:Benzoquinones; D007506:Iron-Sulfur Proteins; D011809:Quinones; C037989:Rieske iron-sulfur protein; C032456:antimycin; C025232:semiquinone radicals; D006418:Heme; D000968:Antimycin A; D014450:Electron Transport Complex III