LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF A HIGHLY-ACTIVE 4-SUBUNIT CYTOCHROME BC1 COMPLEX FROM RHODOBACTER-SPHAEROIDES

A highly active, large-scale preparation of ubiquinolxytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enyzme 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 it consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250–350 (μmol of cyt c reduced)•(μmol 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 MT 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. The pure enzyme was characterized by a variety of biochemical and biophysical methods, and values for ubiquinol, phospholipid, and iron content were determined. The quinolxytochrome c reductase activity was stimulated by dodecyl maltoside and soybean lecithin and was inhibited in the presence of Triton X-100. Full visible spectrum redox titrations showed the presence of two thermodynamically distinct b hemes and cytochrome c1 with spectral properties and Em values similar to those found in chromatophores. Midpoint potentials of +280 and +325 mV were observed for the Rieske iron-sulfur cluster in the absence and presence of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, respectively, by use of EPR spectroscopy. The Em7 for the Rieske cluster was shifted to a value greater than +450 mV when stigmatellin was added. The redox properties of the antimycin-sensitive ubisemiquinone species were also studied by EPR spectroscopy, and a pH dependence of−53 mV/pH unit was determined for the Em of the bound ubiquinol/ubiquinone couple. © 1990, American Chemical Society. All rights reserved.