Investigation of the Essential Boundary Layer Phospholipids of Cytochrome C Oxidase Using Triton X-100 Delipidation

Beef heart cytochrome c oxidase was initially delipidated by incubation of the complex in 5% Triton X-100 followed by separation of the resulting detergent-protein complex from the detergent-lipid mixed micelles by sedimentation through a glycerol gradient containing 1% Triton X-100. After this treatment, the complex contained 2-3 mol of diphosphatidylglycerol (DPG) per heme aa₃. Further.delipidation could be achieved by a second 5% Triton X-100 incubation and a second glycerol gradient. After the second Triton X-100 treatment, the complex contained only 1-2 mol of DPG per heme aa₃. The electron transport activity of the resulting delipidated complex was a function of the amount of bound DPG. Complex containing 2-3 molecules of DPG per heme aa₃ was fully active in Tween 80 or Tween 20 while enzyme containing a single molecule of DPG retained only 60% of the original electron transport activity when assayed in either Tween 80 or Tween 20. The original activity of this maximally delipidated complex could be regenerated by its incubation with 1% Triton X-100 containing 0.02% DPG but not by a similar incubation in either phosphatidylcholine or phosphatidylethanolamine. These results are interpreted in terms of three classes of boundary layer phospholipids that have different affinities for the enzyme. The one with the lowest affinity is nonessential for activity and can be replaced by a variety of exogenous phospholipids and detergents. The second class is more tightly bound to the enzyme, requiring high concentrations of Triton X-100 for its removal, and is also nonessential for activity. The third class has the highest affinity for cytochrome c oxidase and is comprised of two to three molecules of DPG that are either tightly bound at the enzyme surface or buried in the cytochrome c oxidase complex. These DPG molecules are essential for the maximal activity of the complex and cannot be replaced by exogenous phospholipids and detergents, other than DPG, without loss of activity.