An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives.
The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the  cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might  be useful probes for the interaction of cytochrome c with intact mitochondria.
D001120:Arginine; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism; D004140:Dinitrophenols; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005463:Fluoroacetates; D007700:Kinetics; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium; D010446:Peptide Fragments / Q000032:analysis; D010743:Phospholipids; D011485:Protein Binding; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D014269:Trifluoroacetic Acid
D003574:Cytochrome c Group; D004140:Dinitrophenols; D005463:Fluoroacetates; D010446:Peptide Fragments; D010743:Phospholipids; D001120:Arginine; D014269:Trifluoroacetic Acid; D013385:Succinate Dehydrogenase; D003576:Electron Transport Complex IV; D008239:Lysine