Membrane location of spin-labeled apocytochrome c and cytochrome c determined by paramagnetic relaxation agents.
The mitochondrial precursor protein horse heart apocytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region, and the mature protein yeast cytochrome c was similarly labeled on the single free cysteine residue at position 102 at the C-terminal. The proteins were bound to negatively charged phospholipid bilayers, and the accessibility of the spin-labeled cysteine residues to lipid-soluble molecular oxygen and to the lipid-impermeant chromium oxalate anion was determined from the saturation properties of the ESR spectra. Binding of the protein was found to have a considerable effect on the local oxygen concentrations within the lipid bilayer. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled either in the headgroup or at positions in the sn-2 acyl chain, in the presence of unlabeled protein, identify the position of the spin-labeled cysteine residues in the phospholipid bilayer. The spin label on apocytochrome c bound to phosphatidylglycerol bilayers lies between the 5- and 14-C positions of the lipid acyl chain. Admixture of > or = 75 mol % phosphatidylcholine induces an additional surface-associated apocytochrome c population. The spin label on native and heat-denatured cytochrome c is located at the membrane surface. These different extents of membrane penetration correlate also with the reduction in local oxygen concentration experienced by spin-labeled phospholipids on binding of apo- and holocytochrome c. The possible biological implications of the data are discussed.
Snel
M M
MM
Abteilung Spektroskopie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany.
de Kruijff
B
B
Marsh
D
D
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006736:Horses; D007700:Kinetics; D008051:Lipid Bilayers; D008433:Mathematics; D008929:Mitochondria, Heart / Q000378:metabolism; D010743:Phospholipids; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013113:Spin Labels; D013329:Structure-Activity Relationship
D001059:Apoproteins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010743:Phospholipids; D013113:Spin Labels; D045304:Cytochromes c