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J. Biol. Chem., Vol. 279, Issue 17, 18054-18062, April 23, 2004

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Formation of Protein Tyrosine ortho-Semiquinone Radical and Nitrotyrosine from Cytochrome c-derived Tyrosyl Radical^*

Yeong-Renn Chen, Chwen-Lih Chen, Weiguo Chen, Jay L. Zweier, Ohara Augusto¶, Rafael Radi||, and Ronald P. Mason**

From the Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210, the ^¶Departamento de Bioquímica, Instituto de Química, University of São Paulo, São Paulo, SP 05513-970, Brazil, the ^||Department of Biochemistry and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay, and the ^**Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

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 H₂O₂ revealed a single-line signal with a line width of 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 Zn²⁺, 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 H₂O₂ as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H₂O₂ 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 H₂O₂ 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, H₂O₂, NO, and apoptosis.

Received for publication, July 16, 2003 , and in revised form, February 2, 2004.

^* This work was supported by National Institutes of Health Grants K22-ES11031 (to Y.-R. C.) and RO1s-HL38324, HL63744, and HL65608 (to J. L. Z.) and American Heart Association Beginning Grant-in-aid 0365282B (to Y.-R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 607 Davis Heart & Lung Research Institute, The Ohio State University, 473 W. 12th Ave., Columbus, OH 43210. Tel.: 614-688-4054; Fax: 614-292-8778; E-mail: chen-12{at}medctr.osu.edu.

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