Journal article
Cytochrome c-Included Increase of Motionally Restricted Lipid in Reconstituted Cytochrome c Oxidase Membranes, Revealed by Spin-Label ESR Spectroscopy

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Kleinschmidt, J.; Powell, G.; Marsh, D.
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Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted in bilayer membranes of the anionic lipid dimyristoylphosphatidylglycerol (DMPG) with varying enzyme/DMPG ratio. Lipid-protein interactions in the reconstituted membrane complexes were studied in the presence and absence of saturating amounts of bound cytochrome c, by both chemical binding assays and spin-label ESR spectroscopy. The ESR spectra from a phosphatidylglycerol probe spin-labeled on C-14 of the sn-2 chain revealed two distinct lipid populations differing in their rotational mobility. The stoichiometry of lipids that were restricted in their rotational motion by direct interaction with the integral protein was 50-60 lipids/cytochrome c oxidase monomer, in the absence of cytochrome c, independent of the total lipid/protein ratio. Cytochrome c alone 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 degreesC). However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking place in the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of up to 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturating amounts of cytochrome c were bound. Under these conditions, cytochrome c binds to approximately 9 negatively charged DMPG molecules, independent of the cytochrome c oxidase content in the reconstituted system. A likely explanation for these results is that the surface binding of cytochrome c propagates the motional restriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creating microscopic in-plane domains.

Last updated on 2019-25-07 at 10:19