Nonlinear Electron Paramagnetic Resonance Studies of the Interaction of Cytochrome c Oxidase with Spin-Labeled Lipids in Gel-Phase Membranes

Abstract

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.Cytochrome c oxidase is the terminal member of the mitochondrial respiratory chain that effects the reduction of molecular oxygen to water by means of electrons donated from reduced cytochrome c. The reaction involves pumping protons toward the cytosol to provide the chemiosmotic driving force for ATP synthesis. The cytochrome c oxidase enzyme from beef heart is a multisubunit polytopic transmembrane protein, which contains several metal-ion centers that are designated heme a, heme a 3 , Cu A , and Cu B . The crystal structure of the whole 13-subunit assembly of the bovine enzyme has been determined (1) and further refined recently for different redox forms (2).As a representative large integral membrane protein, cytochrome c oxidase has been the subject of several studies of lipid-protein interactions (3-6) by spin-label EPR 1 methods. The latter has proved to be particularly useful for quantitating the lipid-protein interactions with a wide variety of intrinsic proteins in fluid-phase membranes (for a recent review see ref 7). Far less information is available on lipidprotein interactions in gel-phase lipid membranes. These not only are found in reconstituted systems but also may be considered as generally representative of membrane regions with high lipid packing densities, e.g., in spatially segregated membrane domains (8). The few magnetic resonance studies that concentrate on lipid-protein interactions in gel-phase membranes include 2 H NMR studies of rhodopsin (9) and saturation-transfer electron paramagnetic resonance (ST-EPR) of the myelin proteolipid protein (10) in reconstituted membrane systems. Nonlinear EPR methods (ST-EPR and also progressive saturation EPR) are well suited to such investigations with spin-labeled lipids. This is because both the gel-phase bilayer membrane lipids and the lipids interacting directly with the intramemb...

Electron spin resonance in membrane research: Protein–lipid interactions

“…More sensitive estimation of the exchange rate than is possible from slow-exchange lineshapes comes from ESR saturation experiments because these are optimally matched in time-scale to lipid exchange processes [118][119][120][121]. The increase in spin-lattice relaxation rate of the protein-associated lipids that arises from exchange with the fluid lipid population is given by [122]:…”

“…Although exchange rates are too slow to be measured in lipid gel phases, saturation measurements are able to study the stoichiometry and selectivity of lipid-protein interactions in gel-phase membranes, for which two-components are not easily resolved in the conventional ESR spectra [119,130,131].…”

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

“…More sensitive determination of the exchange rate comes from EPR saturation experiments because these depend on longitudinal (i.e., spin-lattice) relaxation rates, which are optimally matched in timescale to lipid exchange processes (Páli et al 1999, 2000; Arora and Marsh 1998; Arora et al 1999). …”