Lipid-protein interactions in cytochrome c oxidase. A comparison of covalently attached phospholipid photo-spin-label with label free to diffuse in the bilayer.

The aim of this study was to clarify the possible origins of the motion-restricted electron spin resonance (ESR) spectral component observed in membranes. For this purpose, a phospholipid photo-spin-label was synthesized, characterized, and used to study lipid-protein interactions in beef heart cytochrome c oxidase. The probe was designed with a nitroaryl azide incorporated in the phospholipid head group, and a spin-label on the sn-2 side chain, and was radiolabeled. The resulting molecule, 1-palmitoyl-2-(14-proxyl [2-3H]stearoyl)-sn-glycero-3-phospho-N-(4-azido-3-nitrophenyl)ethanolami ne, was stable under subdued light and during the procedures required to reconstitute cytochrome c oxidase in phospholipid bilayers. Upon photolysis, the photo-spin-label reacted with the protein in high yields (50% attached). There was no detectable destruction of the spin-label. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytochrome c oxidase after reaction with the photo-spin-label showed highest levels of attachment to bands I, III, and VII, with some labeling of other bands. The labeling pattern demonstrated a distribution of attachment sites, which was needed for the spin-labeling studies. ESR spectra of the attached labels at 25 degrees C indicated a constant fraction of motion-restricted lipid chains, independent of the lipid to protein ratio. In contrast, a spin-labeled phosphatidylcholine and the prephotolyzed photo-spin-label, both free to diffuse in the bilayer, exhibited behavior in agreement with the multiple equilibria binding model. These results, as well as data obtained with membranes frozen at -196 degrees C, show how several situations that lead to a motion-restricted ESR line shape can be distinguished. This study provides additional evidence that the fraction of lipids normally in contact with protein, and not aggregation artifacts, accounts for the observed motion-restricted component of ESR spectra of reconstituted cytochrome c oxidase in phospholipid bilayers.