INTERACTION OF SPIN-LABELED APOCYTOCHROME-C AND SPIN-LABELED CYTOCHROME-C WITH NEGATIVELY CHARGED LIPIDS STUDIED BY ELECTRON-SPIN-RESONANCE

Apocytochrome c has been spin-labeled with a nitroxide derivative of m aleimide on a cysteine residue at either position 14 or position 17 in the N-terminus. Yeast cytochrome c was spin-labeled with the same mal eimide derivative on its single free cysteine residue at position 102 in the C-terminus. The ESR spectra of spin-labeled apocytochrome c hav e been characterized in different environments with respect both to th e conformation of the protein and to its association with lipid. In bu ffer, the spectrum of spin-labeled apocytochrome c indicates high mobi lity, characteristic of the unfolded structure of the apoprotein, and that of spin-labeled cytochrome c is only slightly less mobile, sugges ting that the site labeled is situated at the surface of the folded ho loprotein. Upon binding the spin-labeled protein to negatively charged lipid membranes composed of dioleoylphosphatidylglycerol (DOPG), the ESR spectra of apocytochrome c evidence a large reduction in the mobil ity of the spin-label group, as also do those of yeast cytochrome c. I n the case of apocytochrome c, this immobilization most likely arises from both an increase in secondary structure and a partial penetration of the protein into the lipid bilayer, in addition to the electrostat ic interaction with the lipid headgroups, whereas for cytochrome c the immobilization observed arises primarily from an intimate association with the membrane surface. When the spin-labeled holocytochrome c is denatured by heating and is bound to DOPG bilayer membranes, a rather mobile ESR spectrum is observed, which demonstrates that the spin-labe l is located at the surface of the membrane in this case. The ESR spec tra of spin-labeled apocytochrome c bound to mixed bilayers of dimyris toylphosphatidylglycerol and dimyristoylphosphatidylcholine (DMPC) con sist of both an immobile and a mobile component. The proportion of the mobile component is increased by increasing the mole fraction of the zwitterionic DMPC in the mixed bilayers. The mobile component represen ts a localization of apocytochrome c at the membrane surface, whereas the immobile component most probably represents the penetration of the precursor protein into the membrane interior. The immobile component assigned to membrane penetration of the precursor protein is still pre sent at negatively charged lipid contents comparable to those in the n ative mitochondrial system. The results are discussed in relation to t he conformation of apocytochrome c, its interaction with lipid, and th e import of the apoprotein into mitochondria.