Identification and extent of fluid bilayer regions in membranous cytochrome oxidase

Abstract

Membranous cytochrome oxidase isolated from mitochondria provides a useful model membrane for studies of hydrophobic lipid association with a fairly well characterized functional protein complex. In order to examine the lipid environments, isotropic and oriented samples of cytochrome oxidase of varying phospholipid content were spin labeled with 5-doxylstearic acid (the 4′,4′-dimethyloxazolidine- N-oxyl derivative of 5-ketostearic acid), and examined by ESR spectroscopy. The isotropic samples exhibit two spectral components. Based on the relative intensity of the two components, the mobility, and the response to hydration, the two components are interpreted as arising from two lipid environments: (1) a layer of lipid bound to the hydrophobic protein surface (boundary lipid) and (2) fluid phospholipid bilayer regions. The extent of the bilayer regions is estimated from integration of the two components of the ESR spectra for samples with different phospholipid/protein ratios. The anisotropy of macroscopically oriented membranous cytochrome oxidase samples containing 0.33 and 0.49 mg phospholipid/mg protein confirms the presence of phospholipd bilayer regions. A spectral analysis reveals a remarkable similarity between the phospholipid bilayer regions in the cytochrome oxidase model membranes and bilayers formed by the isolated lipids, whereas the boundary lipid component has no counterpart in lipid bilayers.