An enzyme kinetics and fluorine-19 nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives

Abstract

The reaction of [horse heart] 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 4 different derivatives with well-resolved 19F NMR spectra were obtained. Peptide mapping techniques indicated that 1 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) while the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a 3-fold greater Km in the cytochrome oxidase reaction. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme crevice region, and that Lys-25 is important in the binding. 19F MNR 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.