Large-scale purification and characterization of a highly active four-subunit cytochrome bc1 complex from Rhodobacter sphaeroides

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Abstract
A highly active, large-scale preparation of unbiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Hodobacter sphaeroides. The enzyme was solubilized form 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 (.mu.mol of cyt c reduced).cntdot.(.mu.mol of cyt c1)-1.cntdot.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., and 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 aminoacid 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 quinol:cytochrome 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.