Cytochrome c-Induced Increase of Motionally Restricted Lipid in Reconstituted Cytochrome c Oxidase Membranes, Revealed by Spin-Label ESR Spectroscopy
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- 作者：Jö ; rg H. Kleinschmidt ; Gary L. Powell ; and Derek Marsh
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：August 18, 1998
- 年：1998
- 卷：37
- 期：33
- 页码：11579 - 11585
- 全文大小：110K
- 年卷期：v.37,no.33(August 18, 1998)
- ISSN：1520-4995
文摘
Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted in bilayermembranes of the anionic lipid dimyristoylphosphatidylglycerol (DMPG) with varying enzyme/DMPGratio. Lipid-protein interactions in the reconstituted membrane complexes were studied in the presenceand 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 ofthe sn-2 chain revealed two distinct lipid populations differing in their rotational mobility. The stoichiometryof lipids that were restricted in their rotational motion by direct interaction with the integral protein was50-60 lipids/cytochrome c oxidase monomer, in the absence of cytochrome c, independent of the totallipid/protein ratio. Cytochrome c alone did not induce a motionally restricted population in the lipidESR spectra, when bound to bilayers of negatively charged DMPG alone, in the fluid phase (at 36 
C).However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking placein the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of upto 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturatingamounts of cytochrome c were bound. Under these conditions, cytochrome c binds to ~9 negativelycharged 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 motionalrestriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creatingmicroscopic in-plane domains.
C).However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking placein the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of upto 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturatingamounts of cytochrome c were bound. Under these conditions, cytochrome c binds to ~9 negativelycharged 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 motionalrestriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creatingmicroscopic in-plane domains.