Abstract
The reaction of horse heart cytochrome c with hydrogen peroxide was investigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule. The intensity of the ESR spectrum corresponding to the DBNBS/*cytochrome c radical adduct was greatly enhanced by performing the reaction under anaerobic conditions, which suggested that the spin trap was competing with O2 for reaction with the radical site(s). Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra. In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/tyrosyl adduct. Carbon-13 labeling of the aromatic ring positions of tyrosine yielded additional hyperfine coupling, demonstrating that the radical site was definitely located on the ring of tyrosine. Mass spectrometry detected as many as four DBNBS/.cytochrome c-derived adducts from the reaction of cytochrome with H2O2. Thus, it would appear four radical sites are formed during the reaction, at least one of which is tyrosine.
Highlights
The reaction of horse heart cytochrome c with hydro- such as cytochrome c, produces highly reactive ferryl-heme gen peroxide was investigated using the ESR spin-trap- species that are capable of oxidizing biomolecules and initiatping technique and the nitroso spin traps 3,5-dibromo- ing lipid peroxidation (8 –12)
When the MNP/1⁄7cyt c-derived adduct was submitted to nonspecific proteolysis, an isotropic three-line spectrum was detected with a hyperfine coupling constant of 15.5 G (Fig. 1C)
An immobilized nitroxide was detected when either DBNBS or MNP was added to the reaction mixture containing cytochrome c and hydrogen peroxide
Summary
15498 –15503, 1996 Printed in U.S.A. ESR Spin-trapping of a Protein-derived Tyrosyl Radical from the Reaction of Cytochrome c with Hydrogen Peroxide*. A high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. Because the reactions of both metmyoglobin and cytochrome c with hydrogen peroxide yield oxidizing species that initiate lipid peroxidation and oxidize organic substrates (8 –14), we have utilized the ESR spin-trapping technique to determine if protein-centered radicals are formed on perfine coupling, demonstrating that the radical site cytochrome c following its reaction with hydrogen peroxide. Mass spectrometry detected as many as four DBNBS/1⁄7cytochrome c-derived adducts from the reaction of cytochrome with H2O2 It would appear four radical sites are formed during the reaction, at least one of which is tyrosine
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