Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents

The mitochondrial protein horse heart cytochrome c was specifically spin-la beled with succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectivel y. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled prote in was bound to negatively charged phospholipid membranes composed of diole oylphosphatidylglycerol, and the accessibility of the spin-labeled lysine r esidues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtaine d for phospholipids spin-labeled in the headgroup region, in the presence o f unlabeled protein, identify the position of the spin-labeled lysine resid ues relative to the phospholipid bilayer surface. We have found that cytoch rome c does not penetrate into the membrane interior and that the active si de of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.