Publisher Summary Bovine Complex III is a dimeric complex with a protein mass of 486 kDa. The isolated dimeric complex III contains 16–18 molecules of tightly bound cardiolipin, 20–40 molecules of phosphatitylethanolamine, 10–20 molecules of phosphatidylcholine, and additionally about 140 molecules of bound Triton X-100. The total molecular mass therefore is around 650 kDa. The structure of 3D-crystals has been resolved recently. Each monomer contains 11 distinct protein subunits. Complex III was delipidated and one subunit, the 6.4 kDa protein, was removed together with phospholipid. The subcomplex, termed subcomplex Dl (Dehpidated; missing 1 subunit), was used to study the dependence of catalytic activity on phospholipid and/or the missing 6.4 kDa protein subunit. All steps were performed at 4°C to keep proteolytic activities low. Aminocaproic acid (6-aminohexanoic acid; 5 mM), 1 mM EDTA, and 0.1 mM PMSF were used as protease inhibitors. PMSF was added to the buffers shortly before use (from a 0.5 M stock solution in DMSO kept at –20°C).