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J. Biol. Chem., Vol. 280, Issue 45, 37339-37348, November 11, 2005

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Superoxide Generation from Mitochondrial NADH Dehydrogenase Induces Self-inactivation with Specific Protein Radical Formation^*

Yeong-Renn Chen1, Chwen-Lih Chen, Liwen Zhang¶, Kari B. Green-Church¶, and Jay L. Zweier

From the Department of Internal Medicine, Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, the Department of Molecular and Cellular Biochemistry, College of Medicine, and the ^¶Campus Chemical Instrument Center, Proteomics and Mass Spectrometry Facility, The Ohio State University, Columbus, Ohio 43210

Mitochondrial superoxide () production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this production; its mechanism of generation is not known. Therefore, the catalytic function of NDH in the mediation of generation was investigated by EPR spin-trapping. In the presence of NADH, generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased production. Destruction of the cysteine ligands of iron-sulfur clusters decreased generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of generation. production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was -dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys²⁰⁶) and tyrosine (Tyr¹⁷⁷) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly²⁰⁰-Ala-Gly-Ala-Tyr-Ile-Cys²⁰⁶-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys²¹⁹ and Ala¹⁷⁶-Tyr¹⁷⁷-Glu-Ala-Gly-Leu-Ile-Gly-Lys¹⁸⁴, were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.

Received for publication, April 12, 2005 , and in revised form, August 30, 2005.

^* This work was supported by Grants ES11031 (to Y-R. C.) and HL63744, HL65608, and HL38324 (to J. L. Z.) from the National Institutes of Health and AHA Beginning Grantin-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.

¹ To whom correspondence should be addressed: 607 Davis Heart & Lung Research Inst., The Ohio State University, 473 W. 12th Ave. Columbus, OH 43210. Tel.: 614-688-4054; Fax: 614-292-8778; E-mail: yeong-renn.chen{at}osumc.edu.

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