Abstract
Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.
Highlights
Oxidative alteration of mitochondrial cytochrome c has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events
Direct electron spin resonance (ESR) Spectra from the Reaction of Cyt c with Hydrogen Peroxide—As previously demonstrated, a broad singlet ESR spectrum with a line width of ϳ10 G can be detected when cyt c solution is mixed with H2O2 [8]
We confirmed that the detected ESR signal was enhanced when native cyt c was replaced by HOCl-cyt c in the same reaction system (Fig. 1) because of the enhanced peroxidase activity of HOCl-cyt c [9]
Summary
Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. Electrons may be released during steps prior to oxidative phosphorylation and subsequently react with molecular oxygen to form superoxide [3], the first product in a chain of reactive oxygen species production. Protein radical(s) induced by the reaction of cyt c with H2O2 and other oxidants have been detected by the ESR spin-trapping technique [8, 9]. Chen et al [9] have demonstrated that site-specific oxidation at the sixth ligand of cyt c can be induced by hypochlorite, forming a modified cyt c with methionine sulfoxide with an enhanced peroxidase activity, which increases the formation of cyt c-derived tyrosyl radical by a factor of 2–3. A direct broad line ESR spectrum from cyt c has been assigned as a peroxynitrite- or H2O2-induced tyrosyl radical
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