title: Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.
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.
authors.affiliation: Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
authors.forename: E A
authors.initials: EA
authors.lastname: Berry
authors.forename: L S
authors.initials: LS
authors.lastname: Huang
authors.forename: V J
authors.initials: VJ
authors.lastname: DeRose
mesh_terms: D002338:Carotenoids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D003573:Cytochrome b Group / Q000737:chemistry; D003575:Cytochromes c1 / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D010743:Phospholipids / Q000032:analysis; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011198:Solanum tuberosum / Q000201:enzymology*; D012995:Solubility; D013057:Spectrum Analysis; D014451:Ubiquinone / Q000032:analysis
chemical_list: D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D010743:Phospholipids; D010940:Plant Proteins; C020730:cytochrome b566; D014451:Ubiquinone; D002338:Carotenoids; C025355:cytochrome b560; D006418:Heme; D003575:Cytochromes c1; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III