Chapter 25 Analysis of electron transfer and superoxide generation in the cytochrome bc1 complex.

During the electron transfer through the cytochrome bc(1) complex (ubiquinol-cytochrome c oxidoreductase or complex III), protons are translocated across the membrane, and production of superoxide anion radicals (O(2)(-)) is observed. The bc(1) complex is purified from broken mitochondrial preparation prepared from frozen heart muscles by repeated detergent solubilization and salt fractionation. The electron transfer of the purified complex is determined spectrophotometrically. The activity depends on the choice of detergent, protein concentration, and ubiquinol derivatives used. The proton translocation activity of 2H(+)/e(-) is determined in the reconstituted bc(1)-PL vesicles. The O(2)(-) production by bc(1) is determined by measuring the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazol[1,2-1]pyrazin-3-one hydrochloride (MCLA)-O(2)(-) adduct during a single turnover of bc(1) complex, with the Applied Photophysics stopped-flow reaction analyzer SX.18MV, by leaving the excitation light source off and registering the light emission. Production of O(2)(-) by bc(1) is in an inverse relationship to its electron transfer activity. Inactivation of the bc(1) complex by incubating at elevated temperature (37 degrees C) or by treatment with proteinase K results in an increase in O(2)(-)-generating activity to the same level as that of the antimycin A-inhibited complex. These results suggest that the structural integrity of protein subunits is not required for O(2)(-)-generating activity in the bc(1) complex.