Phospholipase Digestion of Bound Cardiolipin Reversibly Inactivates Bovine Cytochrome bc1

Abstract

Phospholipids and tightly bound cardiolipin (CL) can be removed from Tween 20 solubilized bovine cytochrome bc₁ (EC 1.10.2.2) by digestion with Crotalus atrox phospholipase A₂. The resulting CL-free enzyme exhibits all the spectral properties of native cytochrome bc₁, but is completely inactive. Full electron transfer activity is restored by exogenous cardiolipin added in the presence of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), but not by cardiolipin alone or by mixtures of phospholipids lacking cardiolipin. Acidic, nonmitochondrial phospholipids, e.g., monolysocardiolipin or phosphatidylglycerol, partially reactivate CL-free cytochrome bc₁ if they are added together with DOPC and DOPE. Phospholipid removal from the Tween 20 solubilized enzyme, including the tightly bound cardiolipin, does not perturb the environment of either cytochrome b₅₆₂ or b₅₆₆, nor does it cause the autoreduction of cytochrome c₁. Cardiolipin-free cytochrome bc₁ also binds antimycin and myxothiazol normally with the expected red shifts in b₅₆₂ and b₅₆₆, respectively. However, the CL-free enzyme is much less stable than the lipid-rich preparation, i.e., (1) many chromatographic methods perturb both cytochrome b₅₆₆ (manifested by a hypsochromic effect, i.e., blue shift of 1.5−1.7 nm) and cytochrome c₁ (evidenced by autoreduction in the absence of reducing agents); (2) affinity chromatographic purification of the enzyme causes pronounced loss of subunits VII and XI (65−80% decrease) and less significant loss of subunits I, IV, V, and X (20−30% decrease); and (3) high detergent-to-protein ratios result in disassembly of the complex. We conclude that the major role of the phospholipids surrounding cytochrome bc₁, especially cardiolipin, is to stabilize the quaternary structure. In addition, bound cardiolipin has an additional functional role in that it is essential for enzyme activity.