CYTOCHROME C-INDUCED INCREASE OF MOTIONALLY RESTRICTED LIPID IN RECONSTITUTED CYTOCHROME-C-OXIDASE MEMBRANES, REVEALED BY SPIN-LABEL ESR SPECTROSCOPY

Cytochrome c oxidase isolated from beef heart mitochondria was reconst ituted in bilayer membranes of the anionic lipid dimyristoylphosphatid ylglycerol (DMPG) with varying enzyme/DMPG ratio. Lipid-protein intera ctions in the reconstituted membrane complexes were studied in the pre sence and absence of saturating amounts of bound cytochrome c by both chemical binding assays and spin-label ESR spectroscopy. The ESR spect ra from a phosphatidylglycerol probe spin-labeled on C-14 of the sn-2 chain revealed two distinct lipid populations differing in their rotat ional mobility. The stoichiometry of lipids that were restricted in th eir rotational motion by direct interaction with the integral protein was 50-60 lipids/cytochrome c oxidase monomer, in the absence of cytoc hrome c, independent of the total lipid/protein ratio. Cytochrome c al one did not induce a motionally restricted population in the lipid ESR spectra, when bound to bilayers of negatively charged DMPG alone, in the fluid phase (at 36 degrees C). However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DM PG membranes increased on binding cytochrome c, indicating structural/ dynamic changes taking place in the membrane. Depending on the DMPG/cy tochrome c oxidase ratio, apparent stoichiometries of up to 115 motion ally restricted lipid molecules/cytochrome c oxidase monomer were foun d, when saturating amounts of cytochrome c were bound. Under these con ditions, cytochrome c binds to similar to 9 negatively charged DMPG mo lecules, independent of the cytochrome c oxidase content in the recons tituted system. A likely explanation for these results is that the sur face binding of cytochrome c propagates the motional restriction of th e lipid chains beyond the first boundary shell of cytochrome c oxidase , possibly creating microscopic in-plane domains.