NMR and ESR studies of the interactions of cytochrome c with mixed cardiolipin-phosphatidylcholine vesicles

Abstract

Preparation and structural investigations of 1 : 4 cardiolipin-phosphatidylcholine vesicles with bound ferricytochrome $\text{c}$ are reported. Size and homogeneity of the vesicles were characterized by column chromatography, ultrafiltration, gel filtration, electron microscopy and ¹H NMR techniques. It was found that the diameter of the vesicles with and without bound cytochrome $\text{c}$ was approximately 300 Å, that the appearnce of the species was typical for single bilayer structures and that the vesicles were stable over several days. ¹H and ¹³C NMR combined with ESR studies of spin labels covalently bound to cytochrome $\text{c}$ were then used to investigate structural aspects of these systems. In agreement with earlier studies the present experiments showed that cytochrome $\text{c}$ is bound on the lipid bilayer surface mainly by ionic interactions. They further provided evidence that binding of cytochrome $\text{c}$ affects thedynamic behavior of the lipid surface whereas the interior of the bilayer structure is rather insensitive to the protein. As a consequence of cytochrome $\text{c}$ binding the two kinds of lipids are segregated, with cardiolipin preferentially localized in a surface immobilized boundary layer separating the cytochrome $\text{c}$ from the fluid bulk of the lipid. Evidence was obtained from the NMR and ESR studies that the bilayer-bound cytochrome $\text{c}$ has a preferred orientation, with the heme group pointing away from the lipid surface. This indicates some quite striking similarities in the behavior of cytochrome $\text{c}$ bound to 1 : 4 cardiopilin-phosphatidylcholine tidylcholine vesicles and to intact mitochondria.