Figure 25.3.

Generation of ${\text{O}}_2^{•-}$ in intact, antimycin A–inhibited and proteinase K-treated cytochrome bc₁ complexes. (A) Data for the mitochondrial bc₁ complex and (B) for R. sphaeroides four-subunit, wild-type bc₁ complex. The concentrations of bovine and R. sphaeroides bc₁ complexes in solution A were 5 μM. Detailed experimental conditions are given in Methods for determining superoxide generation. To digest subunits of the cyt bc₁ complex, the bc₁ solution was diluted with 50 mM Tris-HCl buffer, pH 7.4, containing 0.01% DM, to a protein concentration of 20 mg/ml and incubated with 0.4 mg/ml of proteinase K at room temperature. The electron transfer activity and superoxide generation activity were followed during the course of incubation. When electron transfer activity was completely lost, the incubated mixture was subjected to SDS-PAGE to confirm the protein digestion and to determine ${\text{O}}_2^{•-}$ production.