Abstract
A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.
Highlights
From the Xelland Molecular Biology Division. and the $Chemical BiodynamicsDivision, Lawrence Berkeley Laboratory, University of California, Berkeley, &ifornia 94720
A procedure is described for isolation of active ubi- Ubiquinol-cytochrome c oxidoreductase (EC 1.10.2.2, the quinol-cytochrome c oxidoreductase (b c l complex) cytochrome bcl complex) is a multi-subunit enzyme complex from potato tuber mitochondria using dodecyl malto- involved in electron transport in energy conserving memside extraction anidon exchange chromatography
While the work reported here was in progress, Peiffer et al [20] applied ion exchange chromatography to dodecyl maltoside extracts of wheat germ mitochondria
Summary
From the Xelland Molecular Biology Division. and the $Chemical BiodynamicsDivision, Lawrence Berkeley Laboratory, University of California, Berkeley, &ifornia 94720. Traditional methods for purification of the bc, complex use bile salt solubilization and ammonium sulfate precipitation. Such procedures have proved quite successful for the enzyme heterogeneity or include a third subunit. With 1-6 mol of phospholipid and about 0.2 mol of Asahi’s group [8] purified the complex from sweet potato ubiquinonelmol of cytochrome cl. It cata- mitochondria using cholate and ammonium sulfate precipilyzes electron transfer from a short chain ubiquinol tation, but lost all enzymatic activity in the process. EPR spectroscopy shows the presence of a Riesketype iron sulfur center, and the absence of centers associated with succinateand NADH dehydrogenases
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